Method of preparing food using rice oil

ABSTRACT

The present invention relates to methods of preparing food using rice oil which has increased levels of oleic acid and decreased palmitic and linoleic acids, for increased stability to oxidation and health benefits.

This application is a divisional of U.S. Ser. No. 12/309,276, a §371national stage of PCT International Application No. PCT/AU2007/000977,filed Jul. 13, 2007, and claims priority of Australian PatentApplication No. 2006903810, filed Jul. 14, 2006, the contents of all ofwhich are hereby incorporated by reference into this application.

FIELD OF THE INVENTION

The present invention relates to rice oil, rice bran and rice seedswhich have altered levels of oleic acid, palmitic acid and/or linoleicacid. The present invention also provides methods for geneticallymodifying rice plants such that rice oil, rice bran and rice seedsproduced therefrom have altered levels of oleic acid, palmitic acidand/or linoleic acid.

BACKGROUND OF THE INVENTION

Plant oils are an important source of dietary fat for humans,representing about 25% of caloric intake in developed countries (Brounet al., 1999). The current world production of plant oils is about 110million tones per year, of which 86% is used for human consumption.Almost all of these oils are obtained from oilseed crops such assoybean, canola, sunflower, cottonseed and groundnut, or plantationtrees such as palm, olive and coconut (Gunstone, 2001; Oil World Annual,2004). The growing scientific understanding and community recognition ofthe impact of the individual fatty acid components of food oils onvarious aspects of human health is motivating the development ofmodified vegetable oils that have improved nutritional value whileretaining the required functionality for various food applications.These modifications require knowledge about the metabolic pathways forplant fatty acid synthesis and genes encoding the enzymes for thesepathways (Liu et al., 2002a; Thelen and

Ohlrogge, 2002).

Considerable attention is being given to the nutritional impact ofvarious fats and oils, in particular the influence of the constituentsof fats and oils on cardiovascular disease, cancer and variousinflammatory conditions. High levels of cholesterol and saturated fattyacids in the diet are thought to increase the risk of heart disease andthis has led to nutritional advice to reduce the consumption ofcholesterol-rich saturated animal fats in favour of cholesterol-freeunsaturated plant oils (Liu et al., 2002a). While dietary intake ofcholesterol present in animal fats can significantly increase the levelsof total cholesterol in the blood, it has also been found that the fattyacids that comprise the fats and oils can themselves have significanteffects on blood serum cholesterol levels. Of particular interest is theeffect of dietary fatty acids on the undesirable low density lipoprotein(LDL) and desirable high density lipoprotein (HDL) forms of cholesterolin the blood. In general, saturated fatty acids, particularly myristicacid (14:0) and palmitic acid (16:0), the principal saturates present inplant oils, have the undesirable property of raising serumLDL-cholesterol levels and consequently increasing the risk ofcardiovascular disease (Zock et al., 1994; Hu at al., 1997). However, ithas become well established that stearic acid (18:0), the other mainsaturate present in plant oils, does not raise LDL-cholesterol, and mayactually lower total cholesterol (Bonanome and Grundy, 1988; Doughertyet al., 1995). Stearic acid is therefore generally considered to be atleast neutral with respect to risk of cardiovascular disease (Tholstrup,et al, 1994). On the other hand, unsaturated fatty acids, such as themonounsaturate oleic acid (18:1) and the polyunsaturates linoleic acid(18:2) and α-linolenic acid (ALA, 18:3), have the beneficial property oflowering LDL-cholesterol (Mensink and Katan, 1989; Roche and Gibney,2000), thus reducing the risk of cardiovascular disease.

Although nutritionally desirable, highly unsaturated oils are toounstable for use in many food applications, particularly for commercialdeep-frying where they are exposed to high temperatures and oxidativeconditions for long periods of time. Under such conditions, theoxidative breakdown of the numerous carbon double bonds present inunsaturated oils results in the development of short-chain aldehyde,hydroperoxide and keto derivatives, imparting undesirable flavours andreducing the frying performance of the oil by raising the level of polarcompounds (Chang et al., 1978; Williams et al., 1999).

Oil processors and food manufacturers have traditionally relied onhydrogenation to reduce the level of unsaturated fatty acids in oils,thereby increasing their oxidative stability in frying applications andalso providing solid fats for use in margarine and shortenings.Hydrogenation is a chemical process that reduces the degree ofunsaturation of oils by converting carbon double bonds into carbonsingle bonds. Complete hydrogenation produces a fully saturated fat.However, the process of partial hydrogenation results in increasedlevels of both saturated fatty acids and monounsaturated fatty acids.Some of the monounsaturates formed during partial hydrogenation are inthe trans isomer form (such as elaidic acid, a trans isomer of oleicacid) rather than the naturally occurring cis isomer (Sebedio et at,1994; Fernandez San Juan, 1995). In contrast to cis-unsaturated fattyacids, trans-fatty acids are now known to be as potent as palmitic acidin raising serum LDL cholesterol levels (Mensink and Katan, 1990; Noakesand Clifton, 1998) and lowering serum HDL cholesterol (Zock and Katan,1992), and thus contribute to increased risk of cardiovascular disease(Ascherio and Willett, 1997). As a result of increased awareness of theanti-nutritional effects of trans-fatty acids, there is now a growingtrend away from the use of hydrogenated oils in the food industry, infavour of fats and oils that are both nutritionally beneficial and canprovide the required functionality without hydrogenation, in particularthose that are rich in either oleic acid where liquid oils are requiredor stearic acid where a solid or semi-solid fat is preferred.

Plant oils are composed almost entirely of triacylglycerols (TAG)molecules, which consist of three fatty acid (acyl) chains esterified toa glycerol backbone and are deposited in specialised oil body structurescalled oleosomes (Stymne and Stobart, 1987). These storage lipids serveas an energy source for the germinating seedling until it is able tophotosynthesise. Edible plant oils in common use are generally comprisedof five main fatty acids—the saturated palmitic and stearic acids, themonounsaturated oleic acid, and the polyunsaturated linoleic andα-linolenic acids. In addition to fatty acids, plant oils also containsome important minor components such as tocopherols, phytosterols,terpenes and mixed isoprenoids. These minor constituents are ofincreasing interest because some have been shown to exert beneficialeffects on skin health, aging, eyesight and blood cholesterol orpreventing breast cancer or cardiovascular disease (Theriault et al.,1999; Moghadasian and Frohlich, 1999).

Fatty Acid and TAG Synthesis in Seeds

A diagrammatic overview of the metabolic pathways for fatty acidssynthesis in developing seeds is shown in FIG. 1. The initial stages offatty acid synthesis occur in the plastid compartments of the cell,where synthesis of fatty acid carbon chains is initiated with a C2molecule and extended through a stepwise condensation process wherebyadditional C2 carbon units are donated from malonyl-ACP to theelongating acyl chains. The first step in this sequence involvesacetyl-CoA condensing with malonyl-ACP and is catalysed by theβ-ketoacyl synthase III (KASIII) enzyme. The subsequent condensationrounds are catalysed by β-ketoacyl synthase I (KASI) and result in theeventual formation of a saturated C16 acyl chain joined to acyl carrierprotein (ACP), palmitoyl-ACP. The final elongation within the plastid iscatalysed by β-ketoacyl synthase II (KASII) to form the saturated C18acyl chain, stearoyl-ACP. When desaturation occurs, the first doublebond is introduced into the Δ9 position of the C18 chain by a solubleenzyme in the plastid, stearoyl-ACP Δ9-desaturase, to yield themonounsaturated C18:1 oleoyl-ACP.

Fatty acids thus synthesised are either retained in the plastid forfurther modification and incorporation into plastidic lipids, or arereleased from their ACPs by acyl-thioesterases to produce free fattyacids which are exported into the cytosol for further modification andeventual incorporation into TAG molecules. Higher plants have been foundto have at least two types of acyl-thioesterase, FatA with substratespecificity towards oleoyl-ACP, an unsaturated acyl-ACP, and FatB withpreference for saturated acyl-ACPs (Jones at al., 1995; Voelker at al.,1996).

On exiting the plastids, free fatty acids become esterified to Co-enzymeA (CoA) and are then available for transfer to glycerol 3-phosphate(G-3-P) backbones to form lysophosphatidic acid (LPA), phosphatidic acid(PA) and phosphatidyl-choline (PC). Additionally, in some plants,notably the Brassica species, oleic acid esterified to CoA is able to beelongated to form eicosenoic acid (C20:1) and erucic acid (C22:1). Oleicacid esterified to PC is available for further modification beforeincorporation into TAG. In edible oils, the principal modifications onPC are the sequential desaturations of oleic acid to form linoleic andα-linolenic acids by the microsomal Δ12-desaturase (Fad2) andΔ15-desaturase (Fad3) enzymes respectively.

Modification of Existing Fatty Acid Biosynthetic Enzymes

Gene inactivation approaches such as post transcriptional gene silencing(PTGS) have been successfully applied to inactivate fatty acidbiosynthetic genes and develop nutritionally improved plant oils inoilseed crops. For example, soybean lines with 80% oleic acid in theirseed oil were created by cosuppression of the Fad2 encoded microsomalΔ12-desaturase (Kinney, 1996). This reduced the level ofΔ12-desaturation and resulted in accumulation of high amounts of oleicacid. Using a similar approach, cosuppression-based silencing of theFad2 gene was used to raise oleic acid levels in Brassica napus and B.juncea (Stoutjesdijk at al., 2000). Likewise, transgenic expression incottonseed of a mutant allele of the Fad2 gene obtained from rapeseedwas found to be able to suppress the expression of the endogenous cottonFad2 gene and resulted in elevated oleic acid content in about half ofthe primary transgenic cotton lines (Chapman et al., 2001). In anothervariation, transgenic expression in soybean of a Fad2 gene terminated bya self-cleaving ribozyme was able to inactivate the endogenous Fad2 generesulting in increased oleic acid levels (Buhr et al., 2002).RNAi-mediated gene silencing techniques have also been employed todevelop oilseeds with nutritionally-improved fatty acid composition. Incottonseed, transgenic expression of a hairpin RNA (hpRNA) genesilencing construct targeted against ghFad2-1, a seed-specific member ofcotton Fad2 gene family, resulted in the increase of oleic acid fromnormal levels of 15% up to 77% of total fatty acids in the oil (Liu etal., 2002b). This increase was mainly at the expense of linoleic acidwhich was reduced from normal levels of 60% down to as low as 4%.

Fatty Acids in Cereals

In contrast to the considerable work done on fatty acid biosynthesis andmodification in oilseeds, oil modification in cereals is relativelyunexplored. This is probably due to the much lower levels of oils (about1.5-6% by weight) in cereal grains and consequently the perceived lowerimportance of oils from cereals in the human diet.

Rice (Oryza sativa L.) is the most important cereal crop in thedeveloping world and is grown widely, particularly in Asia whichproduces about 90% of the world total. The vast majority of rice in theworld is eaten as “white rice” which is essentially the endosperm of therice grain, having been produced by milling of harvested grain to removethe outer bran layer and germ (embryo and scutellum). This is doneprimarily because “brown rice” does not keep well on storage,particularly under hot tropical conditions.

The oil content of cereal grains such as rice (4%) is quite low relativeto oilseeds where oil can make up to 60% of the weight of the grain(Ohlrogge and Jaworski, 1997). However, lipids may still comprise up to37% of the dry weight of the cereal embryo (Choudhury and Juliano 1980).Most of the lipid content in the rice grain is found in the outer branlayer (Resurreccion et al, 1979) but some is also present in theendosperm, at least some associated with the starch (Tables 1 and 2).The main fatty acids in rice oil are palmitic (16:0) (about 20% of totalfatty acids in the TAG), oleic (18:1) (about 40%), and linoleic acids(18:2) (about 34%) (Radcliffe et at, 1997). There is a range of levelsnaturally occurring in different rice cultivars, for example for oleicacid, from 37.9% to 47.5% and for linoleic (18:2), from 38.2% to 30.4%(Taira et al., 1988).

TABLE 1 Typical fatty acid composition (wt % of total fatty acids) forselected fatty acids of various plant oils. Plant 16:0 18:1 18:2 Barley18 22 54 Soybean 11 23 51 Peanut 8 50 36 Canola 4 63 20 Olive 15 75 9Rice bran 22 38 34

While the FatB gene has been shown to have a high affinity to catalysingthe production of free palmitic acid which is subsequently converted topalmitoyl-CoA in oilseed plants and dicot plants, no information isreported on the role of FatB in rice or other cereals.

TABLE 2 Fatty acid composition (wt % of total fatty acids) for selectedfatty acids of plant lipids associated with starch. Plant 16:0 18:1 18:2Wheat 35-44 6-14 42-52 Barley 55  4 36 Rye 23 41 35 Oat 40 22 35 Maize37 11 46 Maize- High amylose 36 20 38 Maize- waxy 36 23 36 Millet 36 2829 Rice 37-48 9-18 29-46 Data adapted from: Morrison (1988).

Some of the fatty acid desaturases have been characterized in rice butnot Fad2. Akagai et al, (1995) published the nucleotide sequence of agene on rice chromosome 4 encoding stearoyl-acyl carrier proteindesaturase from developing seeds. The gene product participates in theproduction of oleoyl ACP from stearoyl ACP. Kodama et al. (1997)reported the structure, chromosomal location and expression of Fad3 inrice.

The proportion of linolenic acid (18:3) in rice seed oil has beenincreased ten-fold by using soybean Fad3 expression (Anal et al., 2003).More recently, there have been reports of the production of conjugatedlinoleic acid in rice by introduction of a linoleate isomerase gene frombacteria (Kohno-Murase et al., 2006); conjugated linoleic acid isreported to have anti-carcinogenic activity. In a similar vein, in vitromodification of rice bran oil to incorporate capric acid, which mayimprove dietary lipid utilisation in some diseases, using immobilizedmicrobial enzymes has also been reported (Jennings and Akoh, 2000). Inmaize, Fad2 and FA-6 desaturase genes have been sequenced and mapped tochromosomes (Mikkilinen and Rocheford, 2003). The Fad2 and Fad6 clonescould not be mapped to any QTLs for oleic/linoliec acid ratios in themaize grain. There are no published reports of other Fad2 or FatB genescharacterized from rice, maize or wheat.

Storage of Rice

Storage of rice for prolonged times at high temperatures impairs grainquality due to hydrolytic and oxidative deterioration of bran oil.Dehulling the outer husk during harvest to produce brown rice disturbsthe outer bran layers, which allows the oil to diffuse to the outerlayers. Endogenous and microbial lipases then catalyse the hydrolysis oftriglycerides to free fatty acids (FFA) which are then oxidised toproduce an off-flavour (Yasumatsu at al., 1966, Tsuzuki at al., 2004,Thou et al., 2002 Champagne and Grimm, 1995).

Hexanal is the major component increased in the headspace of raw andcooked brown rice stored at high temperatures (Boggs et al., 1964,Shibuya et al., 1974, Tsugita et al., 1983). However, hexanal itself isnot the main cause of the ‘off’ smell; the unattractive smell andflavour of deteriorated rice is probably due to a mixture of thevolatiles that are increased after storage. These include alkanals,alkenals, aromatic aldehydes, ketones, 2-pentylfuran, 4-vinylphenol andothers (Tsugita at al., 1983). Nevertheless, hexanal levels duringstorage have been shown to be associated with the oxidation of linoleicacid (18:2) in brown rice and therefore are a good indicator ofoxidative deterioration (Shin at al., 1986).

The production of hexanal from linoleic acid can be catalysed by theenzyme lipoxygenase (LOX) (St Angelo at al., 1980). Suzuki at al. (1999)identified rice varieties lacking Lox3 and found that on storage of themutant grain at 35° C. for 8 weeks, less hexanal was formed in theheadspace vapour, both for raw and cooked brown rice grain. They alsofound that mutant rice formed less pentanal and pentanol.

The nutrient-rich outer rice bran layer obtained through polishing theouter layers of the rice grain is an excellent food source, containingantioxidant compounds such as tocotrienols and gamma-oryzanol which isalso a phytoestrogen (Rukmini and Raghuram, 1991). The bioactivecompounds present in rice bran oil have been found to lower cholesterolin humans (Most at al., 2005). These bioactive components have also beenshown to improve lipid profiles in rats fed a high cholesterol diet (Haat al., 2005). Another important component found primarily in the branis vitamin A precursors. However, these nutritional and health benefitsare lost through the polishing of rice and the consumption of whiterice.

There is still a need for cereal, such as rice, varieties that producegrain with an improved oil composition for health benefits, which at thesame time is more stable on storage, allowing greater use of, forexample, brown rice in the human diet.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides rice oil having a fattyacid composition comprising greater than about 48% oleic acid, less thanabout 17% palmitic acid, less than about 30% linoleic acid and/or anycombination thereof.

In an embodiment, the ratio of oleic acid to linoleic acid is greaterthan 1.5:1, preferably greater than 2:1, more preferably greater than3:1, and even more preferably greater than 4:1.

In another embodiment, the rice oil has a fatty acid compositioncomprising greater than about 48% oleic acid, less than about 17%palmitic acid, and less than about 30% linoleic acid.

In a further embodiment, the rice oil has a fatty acid compositioncomprising greater than about 40% oleic acid, preferably greater thanabout 50% oleic acid, and even more preferably greater than about 60%oleic acid. In an embodiment, the fatty acid composition of the rice oilis in the range 48-80% oleic acid, 6-16% palmitic acid and 10-25%linoleic acid.

In yet another embodiment, the rice oil has a fatty acid compositioncomprising less than about 16% palmitic acid, preferably less than about15% palmitic acid, more preferably less than about 14% palmitic acid,more preferably less than about 13% palmitic acid, and even morepreferably less than about 12% palmitic acid.

In another embodiment, the rice oil has a fatty acid compositioncomprising less than about 25% linoleic acid, preferably less than about20% linoleic acid, even more preferably less than about 15% linoleicacid.

In another aspect the present invention provides rice bran having afatty acid composition comprising greater than about 48% oleic acid,less than about 17% palmitic acid, less than about 30% linoleic acidand/or any combination thereof.

In an embodiment, the ratio of oleic acid to linoleic acid is greaterthan 1.5:1, preferably greater than 2:1, more preferably greater than3:1, and even more preferably greater than 4:1.

In another embodiment, the rice bran has a fatty acid compositioncomprising greater than about 48% oleic acid, less than about 17%palmitic acid, and less than about 30% linoleic acid.

In a further embodiment, the rice bran has a fatty acid compositioncomprising greater than about 40% oleic acid, preferably greater thanabout 50% oleic acid, and even more preferably greater than about 60%oleic acid. In an embodiment, the fatty acid composition of the ricebran is in the range 48-80% oleic acid, 6-16% palmitic acid and 10-25%linoleic acid.

In yet another embodiment, the rice bran has a fatty acid compositioncomprising less than about 16% palmitic acid, preferably less than about15% palmitic acid, more preferably less than about 14% palmitic acid,more preferably less than about 13% palmitic acid, and even morepreferably less than about 12% palmitic acid.

In another embodiment, the rice bran has a fatty acid compositioncomprising less than about 25% linoleic acid, preferably less than about20% linoleic acid, even more preferably less than about 15% linoleicacid.

In a further aspect, the present invention provides a rice seed having afatty acid composition comprising greater than about 48% oleic acid,less than about 17% palmitic acid, leas than about 30% linoleic acidand/or any combination thereof.

In an embodiment, the ratio of oleic acid to linoleic acid is greaterthan 1.5:1, preferably greater than 2:1, more preferably greater than3:1, and even more preferably greater than 4:1.

In another embodiment, the rice seed has a fatty acid compositioncomprising greater than about 48% oleic acid, less than about 17%palmitic acid, and less than about 30% linoleic acid.

In a further embodiment, the rice seed has a fatty acid composition(i.e. of the oil inside the seed) comprising greater than about 40%oleic acid, preferably greater than about 50% oleic acid, and even morepreferably greater than about 60% oleic acid.

In an embodiment, the fatty acid composition of the rice seed is in therange 48-80% oleic acid, 6-16% palmitic acid and 10-25% linoleic acid.

In yet another embodiment, the rice seed has a fatty acid compositioncomprising less than about 16% palmitic acid, preferably less than about15% palmitic acid, more preferably less than about 14% palmitic acid,more preferably less than about 13% palmitic acid, and even morepreferably less than about 12% palmitic acid.

In another embodiment, the rice seed has a fatty acid compositioncomprising less than about 25% linoleic acid, preferably less than about20% linoleic acid, even more preferably less than about 15% linoleicacid.

In any of the embodiments described above for rice bran or rice seed,the fatty acid composition is typically determined by extraction of theoil and analysis by FAME/GC as described in Example 1. Compositions forindividual fatty acids are expressed as percent (w/w) of the total fattyacids in the oil.

In a further aspect, provided is a rice plant which produces rice oil ofthe invention, rice bran of the invention, and/or a rice seed of theinvention.

In another aspect, the present invention provides an isolatedpolynucleotide which, when present in a cell of a rice plant,down-regulates the level of activity of a Fad2 and/or FatB polypeptidein the cell when compared to a cell that lacks said polynucleotide.

Preferably, the polynucleotide is operably linked to a promoter capableof directing expression of the polynucleotide in a cell of a rice plant.

In an embodiment, the polynucleotide down-regulates mRNA levelsexpressed from at least one Fad2 and/or FatB gene.

Examples of suitable polynucleotides include, but are not limited to, apolynucleotide selected from: an antisense polynucleotide, a sensepolynucleotide, a catalytic polynucleotide, a microRNA, a polynucleotidewhich encodes a polypeptide which binds a Fad2 or FatB polypeptide and adouble stranded RNA.

In an embodiment, the polynucleotide is an antisense polynucleotidewhich hybridises under physiological conditions to a polynucleotidecomprising any one or more of the sequence of nucleotides provided asSEQ NOs 5 to 8, 11 to 14 or 19 to 25.

In a further embodiment, the polynucleotide is a catalyticpolynucleotide capable of cleaving a polynucleotide comprising any oneor more of the sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11to 14 or 19 to 25.

In another embodiment, the polynucleotide is a double stranded RNA(dsRNA) molecule comprising an oligonucleotide which comprises at least19 contiguous nucleotides of any one or more of the sequence ofnucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25, whereinthe portion of the molecule that is double stranded is at least 19basepairs in length and comprises said oligonucleotide.

Preferably, the dsRNA is expressed from a single promoter, wherein thestrands of the double stranded portion are linked by a single strandedportion. Examples of the construction of vectors to produce such dsRNAmolecules is provided in Example 5.

In a preferred embodiment, the polynucleotide, or a strand thereof, iscapable of hybridising to a polynucleotide comprising any one or more ofthe sequence of nucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or19 to 25 under stringent conditions.

In a further aspect, the present invention provides a method ofidentifying a polynucleotide which, when present in a cell of a riceplant, down-regulates the level of activity of a Fad2 and/or FatBpolypeptide in the cell when compared to a cell that lacks saidpolynucleotide, the method comprising

i) determining the ability of a candidate polynucleotide todown-regulate the level of activity of a Fad2 and/or FatB polypeptide ina cell, and

ii) selecting a polynucleotide which down-regulated the level ofactivity of a Fad2 and/or FatB polypeptide in the cell.

Step i) can rely on, for example, analysing the amount or enzymaticactivity of a Fad2 and/or FatB polypeptide, or the amount of mRNAencoding a Fad2 and/or FatB polypeptide in the cell. Alternatively, stepi) may comprise analysing the fatty acid content of the cell, or a seedor plant comprising said cell. Preferably, step i) comprises theintroduction of the candidate polynucleotide or a chimeric DNA includinga promoter operably linked to the candidate polynucleotide into a plantcell, more preferably into a rice cell, and even more preferablycomprises the step of regenerating a transgenic plant from the plantcell and the production of seed from the transgenic plant. The candidategene may be one of a collection of candidate genes, at least 2 or 3 innumber. The invention therefore provides for the use of thepolynucleotides of the invention in a screening method.

The polynucleotide can be, but not limited to, an antisensepolynucleotide, a sense polynucleotide, a catalytic polynucleotide, amicroRNA, a polynucleotide which encodes a polypeptide which binds aFad2 or FatB polypeptide and a double stranded RNA.

In an embodiment, the antisense polynucleotide hybridises underphysiological conditions to a polynucleotide comprising any one or moreof the sequence of nucleotides provided as SEQ II) NOs 5 to 8, 11 to 14or 19 to 25.

In another embodiment, the catalytic polynucleotide is capable ofcleaving a polynucleotide comprising any one or more of the sequence ofnucleotides provided as SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25.

In a further embodiment, the double stranded RNA (dsRNA) moleculecomprises an oligonucleotide which comprises at least 19 contiguousnucleotides of any one or more of the sequence of nucleotides providedas SEQ ID NOs 5 to 8, 11 to 14 or 19 to 25, wherein the portion of themolecule that is double stranded is at least 19 basepairs in length andcomprises said oligonucleotide.

Also provided is an isolated polynucleotide identified using a method ofthe invention.

In a further aspect the present invention provides a vector comprisingor encoding a polynucleotide of the invention.

Preferably, the polynucleotide, or sequence encoding the polynucleotide,is operably linked to a promoter. Preferably, the promoter confersexpression of the polynucleotide preferentially in the embryo,endosperm, bran layer and/or seed of a rice plant relative to at leastone other tissue or organ of said plant.

Also provided is a cell comprising the vector of the invention, and/orthe polynucleotide of the invention.

In an embodiment, the polynucleotide or vector was introduced into thecell or a progenitor of the cell.

In a further embodiment, the cell is a rice cell or an Agrobacteriumcell.

Preferably, the polynucleotide is integrated into the genome of thecell.

In another aspect, the present invention provides a rice plantcomprising a cell of the invention.

In yet another aspect, the present invention provides a method ofproducing the cell of the invention, the method comprising the step ofintroducing the polynucleotide of the invention, or a vector of theinvention, into a cell.

Preferably the method further comprises the step of regenerating atransgenic plant from the cell.

Also provided is the use of the polynucleotide of the invention or avector of the invention to produce a recombinant cell.

In yet another aspect, the present invention provides a geneticallymodified rice plant, wherein the plant has decreased expression of apolypeptide having Fad2 and/or FatB activity relative to a correspondingnon-modified plant.

Preferably, the plant has been transformed such that it comprises apolynucleotide of the invention, or a progeny plant thereof whichcomprises said polynucleotide.

In a further aspect the present invention provides a method of producingrice oil of the invention, rice bran of the invention and/or rice seedof the invention, the method comprising exposing a rice plant to anantagonist of a Fad2 or FatB polypeptide.

In another aspect, the present invention provides a method of obtaininga genetically modified rice plant which can be used to produce brownrice seed with an increased storage life when compared to unmodifiedbrown rice seed, the method comprising genetically manipulating theplant such that the activity and/or level of production of a Fad2 and/orFatB polypeptide is reduced in the rice seed when compared to acorresponding plant which produces unmodified brown rice seed.

Preferably, the activity and/or level of production of a Fad2 and/orFatB polypeptide is only reduced in the seed of the plant.

In an embodiment, the activity and/or level of production of a Fad2polypeptide is reduced.

In a further embodiment, the transgenic plant comprises a polynucleotideof the invention or a vector of the invention.

In a further aspect, the present invention provides a geneticallymodified rice plant produced using a method of the invention, or progenythereof.

In a further aspect, the present invention provides a method ofselecting a rice plant which can be used to produce rice oil of theinvention, rice bran according to the invention and/or rice seed of theinvention, the method comprising;

i) screening a mutagenized population of rice seeds or rice plants, and

ii) selecting a seed or plant which is capable of, producing rice oil ofthe invention, rice bran of the invention and/or rice seed of theinvention.

In one embodiment, step i) comprises analysing a mutagenized seed and/orplant for a polypeptide which has Fad2 or FatB activity.

In another embodiment, step i) comprises analysing the sequence and/orexpression levels of a Fad2 or FatB gene of the mutagenized seed and/orplant.

In a further embodiment, step i) comprises analysing the fatty acidcomposition of the oil, bran and/or seed of the mutagenized seed and/orplant.

In a further aspect, the present invention provides a method ofselecting a rice plant which can be used to produce rice oil of theinvention, rice bran of the invention and/or rice seed of the invention,the method comprising

i) analysing the fatty acid content of said rice oil, rice bran and/orseed obtained from a candidate rice plant, and

ii) selecting a rice plant which can be used to produce rice oil of theinvention, rice bran of the invention and/or rice seed of the invention.

In yet a further aspect, the present invention provides a method ofselecting a rice plant which can be used to produce rice oil of theinvention, rice bran of the invention and/or rice seed of the invention,the method comprising

i) analysing a sample from a candidate plant for a polypeptide which hasFad2 or FatB activity, and

ii) selecting a rice plant which can be used to produce rice oil of theinvention, rice bran of the invention and/or rice seed of the inventionbased on the sequence, level of production and/or activity of saidpolypeptide.

In another aspect, the present invention provides a method of selectinga rice plant which can be used to produce rice oil of the invention,rice bran of the invention and/or rice seed of the invention, the methodcomprising

i) analysing the sequence and/or expression levels of a Fad2 or FatBgene of a candidate plant, and

ii) selecting a rice plant which can be used to produce rice oil of theinvention rice bran of the invention and/or rice seed of the inventionbased on the sequence and/or expression levels of said gene.

In yet another aspect the present invention provides a method ofidentifying a rice plant which can be used to produce rice oil of theinvention, rice bran of the invention and/or rice seed of the invention,the method comprising detecting a nucleic acid molecule of the plant,wherein the nucleic acid molecule is linked to, and/or comprises atleast a part of a Fad2 gene and/or FatB gene in the plant.

In a further aspect, the present invention provides a method ofidentifying a rice plant which can be used to produce brown rice seedwith an increased storage life, the method comprising detecting anucleic acid molecule of the plant, wherein the nucleic acid molecule islinked to, and/or comprises at least a part of, a Fad2 gene and/or FatBgene in the plant.

In an embodiment, the above two methods comprise:

i) hybridising a second nucleic acid molecule to said nucleic acidmolecule which is obtained from said plant,ii) optionally hybridising at least one other nucleic acid molecule tosaid nucleic acid molecule which is obtained from said plant; andiii) detecting a product of said hybridising step(s) or the absence of aproduct from said hybridising step(s).

In an embodiment, the second nucleic acid molecule is used as a primerto reverse transcribe or replicate at least a portion of the nucleicacid molecule.

The nucleic acid can be detected using any technique such as, but notlimited to, restriction fragment length polymorphism analysis,amplification fragment length polymorphism analysis, microsatelliteamplification and/or nucleic acid sequencing.

In an embodiment, the method comprises nucleic acid amplification. Inanother embodiment, the method analyses expression levels of the gene.

Also provided is a method of obtaining a rice plant or seed, the methodcomprising;

i) crossing a first parental rice plant which comprises a Fad2 allelewhich confers an increased proportion of oleic acid in oil of the grainof the plant with a second parental rice plant which comprises a FatBallele which confers a decreased proportion of palmitic acid in oil ofthe grain of the plant;ii) screening progeny plants or grain from the cross for the presence ofboth alleles; andiv) selecting a progeny plant or grain comprising both alleles andhaving an increased proportion of oleic acid and a decreased proportionof palmitic acid in oil of the grain of the plant.

In another aspect, the present invention provides a method ofintroducing a Fad2 allele into a rice plant, the method comprising

i) crossing a first parental rice plant with a second parental riceplant, wherein the second plant comprises said allele, andii) backcrossing the progeny of the cross of step i) with plants of thesame genotype as the first parent plant for a sufficient number of timesto produce a plant with a majority of the genotype of the first parentbut comprising said alleleiii) selecting a plant with the majority of the genotype of the firstplant and comprising said allele;

wherein said allele confers an increased proportion of oleic acid inoil, bran and/or seed of the plant

In yet another aspect, the present invention provides a method ofintroducing a FatB allele into a rice plant, the method comprising

i) crossing a first parental rice plant with a second parental riceplant, wherein the second plant comprises said allele, andii) backcrossing the progeny of the cross of step i) with plants of thesame genotype as the first parent plant for a sufficient number of timesto produce a plant with a majority of the genotype of the first parentbut comprising said alleleiii) selecting a plant with the majority of the genotype of the firstplant and comprising said allele;wherein said allele confers a decreased proportion of palmitic acid inoil, bran and/or seed of the plant.

Further, provided is a method of increasing the proportion of oleic acidin oil, bran and/or seed of a rice plant, the method comprisinggenetically manipulating said plant such that the production of a Fad2polypeptide is decreased when compared to a wild-type plant, wherein thepolypeptide has Δ12 desaturase activity.

In yet another aspect, the present invention provides a method ofdecreasing the proportion of palmitic acid in oil, bran and/or seed of arice plant, the method comprising genetically manipulating said plantsuch that the production of a FatB polypeptide is decreased whencompared to a wild-type plant, wherein the polypeptide has (FatB)activity.

Also provided is a rice plant obtained using a method of the invention,or progeny plant thereof.

In a further aspect, the present invention provides rice oil obtainedfrom a plant of the invention.

In a further aspect, the present invention provides rice bran obtainedfrom a plant of the invention.

In a further aspect, the present invention provides rice seed obtainedfrom a plant of the invention.

Further, provided is a method of producing seed, the method comprising;

a) growing a plant of the invention, and

b) harvesting the seed.

In yet another aspect, the present invention provides a food productcomprising rice oil of the invention, rice bran of the invention and/orrice seed of the invention.

In another aspect, the present invention provides a method of preparingfood, the method comprising cooking an edible substance in rice oil ofthe invention.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1—Principal fatty acid biosynthetic pathways in plastid and cytosolof higher plants.

FIGS. 2(A)-(B)—Alignment of rice FatB proteins. ProteinFA1B2=SEQ IDNO:1, proteinFATB3=SEQ ID NO:2, proteinFATB1=SEQ ID NO:3 andproteinFATB4=SEQ ID NO:4.

FIG. 3—Rice FatB gene structures. LOC_Os02g4=SEQ ID NO:5, LOC_Os11g4=SEQID NO:6, LOC_Os06g0=SEQ ID NO:7 and LOC_Os06g3=SEQ ID NO:8.

FIGS. 4(A)-(L)—Alignment of FatB gene sequences by ClustalW. Defaultparameters were used and note that the ‘genes’ are of different lengths.

FIGS. 5(A)-(C)—The exon-intron structures of the FatB gene correspondingto LOC_Os6g05130. The top line corresponds to the start of the codingsequence in mRNA (SEQ ID NO:9) and the second line of the paircorresponds to the gene (SEQ ID NO:10).

FIGS. 6(A)-(B)—Alignment of coding sequence of four FatB isoformsshowing consecutive exons in alternating lower case and upper caserespectively and location of primers (underlined) used to distinguishthe isoforms by RT-PCR. The initiating codon (start position 1) is inbold. ACI08870=SEQ ID NO:1 I, AP005291=SEQ ID NO:12, AP000399=SEQ IDNO:13 and AP004236=SEQ ID NO: 14.

FIGS. 7(A)-(B) Clustal W alignment of deduced polypeptide sequence ofFad2 isoforms. Note that line F_(—)1 corresponds to Os02g48S60, F_(—)2corresponds to Os07g23410, F_(—)3 to Os07g23430 and O_(—)4 toOs07g23390. The program ClustalW (Fast) with default parameters wasused. ProteinF_(—)1=SEQ ID NO:15, ProteinF_(—)3=SEQ ID NO:16,ProteinF_(—)2=SEQ ID NO:17 and ProteinF_(—)4=SEQ ID NO:18,

FIGS. 8(A)-(B)—Alignment of Fad2 sequences showing location of 5′ UTR inisoform AP004047 (lowercase) and location of primers used foramplification by RT-PCR (underlined). The location of the stop codon isindicated by a box and untranslated regions downstream of the stop codonare in lower case. AP005168=SEQ ID NO:19, AP004047=SEQ ID NO:20 andContig2654=SEQ ID NO:21.

FIG. 9—Rice Fad2 gene structures.

FIGS. 10(A)-(D)—Alignment of the nucleotide sequences of the proteincoding regions for rice Fad2 genes. Line 0_(—)2 corresponds toOs07g23410, 0_(—)4 to Os07g23390, 0_(—)1 to Os02g48560 and 0_(—)3 toOs07g23410. The program ClustalW with default parameters was used.CdsFAD20_(—)2=SEQ ID NO:22, CdsFAD20_(—)4=SEQ ID NO:23,CdsFAD20_(—)1=SEQ ID NO:24 and CdsFAD20_(—)3=SEQ ID NO:25.

FIG. 11—Graphical representation of the relative percentages ofpalmitic, oleic and linoleic acid as determined by GC analysis of totaloil fraction from grains of indicated genotypes.

FIG. 12—Graphical representation of the relative percentages of palmiticand oleic acid in a GC total lipid analysis from grains of the genotypesindicated.

FIG. 13—Graphical representation of the relative percentages of linoleicand oleic acid in a GC total lipid analysis from grains of the genotypesindicated.

FIG. 14—Scatterplot showing the percentage of linoleic versus oleic acidin the grain of rice plants of the indicated genotypes. Note that therelationship (reflected in the slope of the line) between the amounts ofthese two fatty acids is essentially the same in all of the linesanalysed but the pool size capacity appears to be different (asreflected in the displacements of the different lines along the spaceanalysed.

FIG. 15—Scatterplot of the percentage of linoleic versus palmitic acidamong different genotypes. Note the different slopes of the linesaffected in FatB compared to those affected in Fad2. This suggests therelationship between these components is different in the differentgenotypes, probably reflecting the differences in the steps affected.

FIG. 16—Scatterplot of the percentage of oleic versus palmitic acid forvarious genotypes. Note the differences in slopes.

FIG. 17—Graphical representation of Principal Component Analysis ofvariation in oil composition for Fad2 RNAi plants and FatB RNAi plants.The results show that Principal Component 2 is Linoleic versus Oleic andPrincipal Component 2 is Palmitic versus Linoleic plus Oleic.

FIG. 18—Western blot showing the reaction of antisera raised againstpeptide. FatB-99 against total leaf protein extracts analysed bySDS-PAGE. A peptide of approx 20 kDa is missing in the Tos-17 line. Thereaction with preimmune sera is indicated. R refers to FatB RNAi linesand T is the Tos-17 line. W and W2 are wildtype.

KEY TO SEQUENCE LISTING

SEQ ID NO:1—Rice FatB2 protein.SEQ ID NO:2—Rice FatB3 protein.SEQ ID NO:3—Rice FatB1 protein.SEQ ID NO:4—Rice FatB4 protein.SEQ ID NO:5—Rice FatB3 gene.SEQ ID NO:6—Rice FatB2 gene.SEQ ID NO:7—Rice FatB1 gene.SEQ ID NO:8—Rice FatB4 gene.SEQ ID NO:9—eDNA encoding rice FatB1 protein.SEQ ID NO:10—Gene encoding rice FatB1 protein (partial sequence only,see FIG. 5—includes all exon sequences and some flanking and intronsequence).SEQ ID NO: 11—Open reading frame encoding rice FatB2 protein.SEQ ID NO:12—Open reading frame encoding rice FatB3 protein.SEQ ID NO: 13—Open reading frame encoding rice FatB1 protein.SEQ ID NO:14—Open reading frame encoding rice FatB4 protein.SEQ ID NO:15—Rice Fad2 isoform 1.SEQ ID NO:16—Rice Fad2 isoform 3.SEQ ID NO:17—Rice Fad2 isoform 2.SEQ ID NO:18—Rice Fad2 isoform 4.SEQ ID NO:19—Rice Fad2-3 cDNA.SEQ ID NO:20—Rice Fad2-1 cDNA.SEQ ID NO:21—Rice Fad2-2 cDNA.SEQ ID NO:22—Open reading frame encoding rice Fad2-2.SEQ ID NO:23—Open reading frame encoding rice Fad2-4.SEQ ID NO:24—Open reading frame encoding rice Fad2-1.SEQ ID NO:25—Open reading frame encoding rice Fad2-3.SEQ ID NO:26—FatB consensus sequence.SEQ ID NOs 27 to 33—Fad2 consensus sequences.

SEQ ID NOs 34 to 55 and 60 to 63—Oligonucleotide

SEQ ID NOs 56 to 59—Antigenic rice FatB peptides.SEQ ID NOs 64 to 83—Sequence of one strand of molecules that can be usedfor RNAi.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,plant molecular biology, molecular genetics, immunology,immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook at al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and S. D. Flames (editors), DNA Cloning: APractical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M.Ausubel at al. (editors), Current Protocols in Molecular Biology, GreenePub. Associates and Wiley-Interscience (1988, including all updatesuntil present), Ed Harlow and David Lane (editors) Antibodies: ALaboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E.Coligan et al. (editors) Current Protocols in Immunology, John Wiley &Sons (including all updates until present).

Selected Definitions

As used herein, the term “Fad2 polypeptide” refers to a protein whichperforms a desaturase reaction converting oleic acid to linoleic acid.Thus, the term “Fad2 activity” refers to the conversion of oleic acid tolinoleic acid. These fatty acids may be in an esterified form, such as,for example, as part of a phospholipid. Examples of rice Fad2polypeptides include proteins comprising an amino acid sequence providedin FIG. 7 and SEQ ID NOs 15 to 18, as well as variants and/or mutantsthereof. Such variants and/or mutants may be at least 80% identical,more preferably at least 90% identical, more preferably at least 95%identical, and even more preferably at least 99% identical to any one ofthe polypeptides provided in FIG. 7 and SEQ ID NOs 15 to 18.

A “Fad2 polynucleotide” or “Fad2 gene” encodes a Fad2 polypeptide.Examples of Fad2 polynucleotides include nucleic acids comprising anucleotide sequence provided in FIG. 8 or 10 and SEQ ID NOs 19 to 25, aswell as allelic variants and/or mutants thereof. Examples of Fad2 genesinclude nucleic acids comprising a nucleotide sequence provided in FIG.8 and SEQ ID NOs 19 to 21, as well as allelic variants and/or mutantsthereof. Such allelic variants and/or mutants may be at least 80%identical, more preferably at least 90% identical, more preferably atleast 95% identical, and even more preferably at least 99% identical toany one of the polynucleotides provided in FIG. 8 and/or 10, and/or SEQID NOs 19 to 25.

As used herein, the term “FatB polypeptide” refers to a protein whichhydrolyses palmitoyl-ACP to produce free palmitic acid. Thus, the term“FatB activity” refers to the hydrolysis of palmitoyl-ACP to producefree palmitic acid. Examples of rice FatB polypeptides include proteinscomprising an amino acid sequence provided FIG. 2 SEQ ID NOs 1 to 4, aswell as variants and/or mutants thereof. Such variants and/or mutantsmay be at least 80% identical, more preferably at least 90% identical,more preferably at least 95% identical, and even more preferably atleast 99% identical to any one of the polypeptides provided in FIG. 2and SEQ ID NOs 1 to 4.

A “FatB polynucleotide” or “FatB gene” encodes a FatB polypeptide.Examples of FatB polynucleotides include nucleic acids comprising anucleotide sequence provided in FIGS. 4 and 6 and SEQ ID NOs 5 to 8 and11 to 14, as well as allelic variants and/or mutants thereof. Examplesof FatB genes include nucleic acids comprising a nucleotide sequenceprovided in FIG. 4 and SEQ ID NOs 5 to 8, as well as allelic variantsand/or mutants thereof. Such allelic variants and/or mutants may be atleast 80% identical, more preferably at least 90% identical, morepreferably at least 95% identical, and even more preferably at least 99%identical to any one of the polynucleotides provided in FIG. 4 and/or 6,and/or SEQ ID NOs 5 to 8 and 11 to 14.

As used herein, the term “rice” refers to any species of the GenusOryza, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. It is preferred that the plantis of a Oryza species which is commercially cultivated such as, forexample, a strain or cultivar or variety of Oryza saliva or suitable forcommercial production of grain.

As used herein, the term “rice oil” refers to a composition obtainedfrom the seed/grain, or a portion thereof such as the bran layer, of arice plant which comprises at least 60% (w/w) lipid. Rice oil istypically a liquid at room temperature. Preferably, the lipid comprisesfatty acids that are at least 6 carbons in length. The fatty acids aretypically in an esterified form, such as for example astriacylglycerols, phospholipid. Rice oil of the invention comprisesoleic acid. Rice oil of the invention may also comprise at least someother fatty acids such as palmitic acid, linoleic acid, myristic acid,stearic acid and/or linolenic acid. The fatty acids may be free fattyacids and/or be found as triacylglycerols (TAGs). In an embodiment, atleast 50%, more preferably at least 70%, more preferably at least 80% ofthe fatty acids in rice oil of the invention be found as TAGs. Rice oilof the invention can form part of the rice grain/seed or portion thereofsuch as the aleurone layer or embryo/scutellum, which together arereferred to as “rice bran”. Alternatively, rice oil of the invention hasbeen extracted from rice grain/seed or rice bran. An example of such anextraction procedure is provided in Example 1. Thus, in an embodiment,“rice oil” of the invention is “substantially purified” or “purified”rice oil that has been separated from one or more other lipids, nucleicacids, polypeptides, or other contaminating molecules with which it isassociated in its native state. It is preferred that the substantiallypurified rice oil is at least 60% free, more preferably at least 75%free, and more preferably at least 90% free from other components withwhich it is naturally associated. In a preferred embodiment, uponextraction the ratio of oleic acid to linoleic acid, palmitic acid tooleic acid and/or palmitic acid to linoleic acid has not beensignificantly altered (for example, no greater than a 5% alteration)when compared to the ratio in the intact seed/grain or bran. In afurther embodiment, the rice oil has not been exposed to a procedure,such as hydrogenation, which may alter the ratio of oleic acid tolinoleic acid, palmitic acid to oleic acid and/or palmitic acid tolinoleic acid when compared to the ratio in the intact seed/grain orbran. Rice oil of the invention may further comprise non-fatty acidmolecules such as, but not limited to, γ-oryzanols and sterols.

Rice oil may be extracted from rice seed or bran by any method known inthe art. This typically involves extraction with nonpolar solvents suchas diethyl ether, petroleum ether, chloroform/methanol or butanolmixtures. Lipids associated with the starch in the grain may beextracted with water-saturated butanol. The rice oil may be “de-gummed”by methods known in the art to remove polysaccharides or treated inother ways to remove contaminants or improve purity, stability orcolour. The triacylglycerols and other esters in the oil may behydrolysed to release free fatty acids, or the oil hydrogenated ortreated chemically or enzymatically as known in the art.

Rice oil after extraction from rice seed or bran typically comprises thegroup of lipids called γ-oryzanols. As used herein, “comprisesγ-oryzanol” refers to the presence of at least 0.1% (w/w) γ-oryzanolcompounds in the oil. The levels of γ-oryzanol in rice oil afterextraction and before removal from the TAG is typically 1.5-3.5% (w/w).The compounds are typically a mixture of steryl and other triterpenylesters of ferulic acid (4-hydroxy-3-methoxy cinnamic acid). Cycloartenylferulate, 24-methylene cycloartanyl fendate and campesteryl ferulate arethe predominant ferulates in oryzanol, with lower levels of β-sitosterylferulate and stigmasteryl ferulate. The presence of γ-oryzanols isthought to help protect consumers of rice oil against chronic diseasessuch as heart disease and cancer and therefore the presence ofγ-oryzanol is advantageous.

As used herein, the term “rice bran” refers to the layer (aleuronelayer) between the inner white rice grain and the outer hull of a riceseed/grain as well as the embryo/scutellum of the grain. The rice branis the primary by product of the polishing of brown rice to producewhite rice.

As used herein, the term “increased storage life” refers to a method ofthe invention producing a seed/grain, which upon harvesting, can bestored as brown rice for an enhanced period of time when compared to,for example, brown rice harvested from a wild type (non-geneticallymodified) rice plant. As described herein, one measure for “increasedstorage life” of brown rice is hexanal production following storage forat least 8 weeks at 40° C. (see Example 8).

The term “plant” includes whole plants, vegetative structures (forexample, leaves, stems), roots, floral organs/structures, seed(including embryo, endosperm, and seed coat), plant tissue (for example,vascular tissue, ground tissue, and the like), cells and progeny of thesame.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a gene construct (“transgene”) not foundin a wild-type plant of the same species, variety or cultivar. A“transgene” as referred to herein has the normal meaning in the art ofbiotechnology and includes a genetic sequence which has been produced oraltered by recombinant DNA or RNA technology and which has beenintroduced into the plant cell. The transgene may include geneticsequences derived from a plant cell. Typically, the transgene has beenintroduced into the plant by human manipulation such as, for example, bytransformation but any method can be used as one of skill in the artrecognizes.

The terms “seed” and “grain” are used interchangeably herein. “Grain”generally refers to mature, harvested grain but can also refer to grainafter imbibition or germination, according to the context. Mature graincommonly has a moisture content of less than about 18-20%.

As used herein, the term “corresponding non-modified plant” refers to awild-type plant. “Wild type”, as used herein, refers to a cell, tissueor plant that has not been modified according to the invention.Wild-type cells, tissue or plants may be used as controls to comparelevels of expression of an exogenous nucleic acid or the extent andnature of trait modification with cells, tissue or plants modified asdescribed herein. Wild-type rice varieties that are suitable as areference standard include Nipponbare.

“Nucleic acid molecule” refers to a oligonucleotide, polynucleotide orany fragment thereof. It may be DNA or RNA of genomic or syntheticorigin, double-stranded or single-stranded, and combined withcarbohydrate, lipids, protein, or other materials to perform aparticular activity defined herein. The terms “nucleic acid molecule”and “polynucleotide” are used herein interchangeably.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty-5, anda gap extension penalty=0.3. Unless stated otherwise, the query sequenceis at least 45 nucleotides in length, and the GAP analysis aligns thetwo sequences over a region of at least 45 nucleotides. Preferably, thequery sequence is at least 150 nucleotides in length, and the GAPanalysis aligns the two sequences over a region of at least 150nucleotides. More preferably, the query sequence is at least 300nucleotides in length and the GAP analysis aligns the two sequences overa region of at least 300 nucleotides. Even more preferably, the GAPanalysis aligns the two sequences over their entire length.

“Oligonucleotides” can be RNA, DNA, or derivatives of either. Althoughthe terms polynucleotide and oligonucleotide have overlapping meaning,oligonucleotide are typically relatively short single strandedmolecules. The minimum size of such oligonucleotides is the sizerequired for the formation of a stable hybrid between an oligonucleotideand a complementary sequence on a target nucleic acid molecule.Preferably, the oligonucleotides are at least 15 nucleotides, morepreferably at least 18 nucleotides, more preferably at least 19nucleotides, more preferably at least 20 nucleotides, even morepreferably at least 25 nucleotides in length.

As used herein, the term “nucleic acid amplification” refers to any invitro method for increasing the number of copies of a nucleic acidmolecule with the use of a DNA polymerase. Nucleic acid amplificationresults in the incorporation of nucleotides into a DNA molecule orprimer thereby forming a new DNA molecule complementary to a DNAtemplate. The newly formed DNA molecule can be used a template tosynthesize additional DNA molecules.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence, such as a polynucleotide definedherein, if it stimulates or modulates the transcription of the codingsequence in an appropriate cell. Generally, promoter transcriptionalregulatory elements that are operably linked to a transcribed sequenceare physically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising the proteincoding region of a structural gene and including sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof at least about 2 kb on either end and which are involved inexpression of the gene. The sequences which are located 5′ of the codingregion and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region which may be interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene which are transcribed intonuclear RNA (hnRNA); introns may contain regulatory elements such asenhancers. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide. The term“gene” includes a synthetic or fusion molecule encoding all or part ofthe proteins of the invention described herein and a complementarynucleotide sequence to any one of the above.

As used herein, the term “genetically linked” or similar refers to amarker locus and a second locus being sufficiently close on a chromosomethat they will be inherited together in more than 50% of meioses, e.g.,not randomly. This definition includes the situation where the markerlocus and second locus form part of the same gene. Furthermore, thisdefinition includes the embodiment where the marker locus comprises apolymorphism that is responsible for the trait of interest (in otherwords the marker locus is directly “linked” or “perfectly linked” to thephenotype). In another embodiment, the marker locus and a second locusare different, yet sufficiently close on a chromosome that they will beinherited together in more than 50% of meioses. The percent ofrecombination observed between genetically linked loci per generation(centimorgans (cM)), will be less than 50. In particular embodiments ofthe invention, genetically linked loci may be 45, 35, 25, 15, 10, 5, 4,3, 2, or I or less cM apart on a chromosome. Preferably, the markers areless than 5 cM apart and most preferably about 0 cM apart.

An “allele” refers to one specific form of a genetic sequence (such as agene) within a cell, an individual plant or within a population, thespecific form differing from other forms of the same gene in thesequence of at least one, and frequently more than one, variant siteswithin the sequence of the gene. The sequences at these variant sitesthat differ between different alleles are termed “variances”,“polymorphisms”, or “mutations”.

A “polymorphism” as used herein denotes a variation in the nucleotidesequence between alleles of the loci of the invention, of differentspecies, cultivars, strains or individuals of a plant. A “polymorphicposition” is a preselected nucleotide position within the sequence ofthe gene. In some cases, genetic polymorphisms are reflected by an aminoacid sequence variation, and thus a polymorphic position can result inlocation of a polymorphism in the amino acid sequence at a predeterminedposition in the sequence of a polypeptide. In other instances, thepolymorphic region may be in a non-polypeptide encoding region of thegene, for example in the promoter region such may influence expressionlevels of the gene. Typical polymorphisms are deletions, insertions orsubstitutions. These can involve a single nucleotide (single nucleotidepolymorphism or SNP) or two or more nucleotides.

The terms “polypeptide” and “protein” are generally used interchangeablyand refer to a single polypeptide chain which may or may not be modifiedby addition of non-amino acid groups. It would be understood that suchpolypeptide chains may associate with other polypeptides or proteins orother molecules such as co-factors. The terms “proteins” and“polypeptides” as used herein also include variants, mutants,modifications, analogous and/or derivatives of the polypeptides of theinvention as described herein.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 25 aminoacids in length, and the GAP analysis aligns the two sequences over aregion of at least 25 amino acids. More preferably, the query sequenceis at least 50 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 50 amino acids. More preferably,the query sequence is at least 100 amino acids in length and the GAPanalysis aligns the two sequences over a region of at least 100 aminoacids. Even more preferably, the query sequence is at least 250 aminoacids in length and the GAP analysis aligns the two sequences over aregion of at least 250 amino acids. Even more preferably, the GAPanalysis aligns the two sequences over their entire length.

Antisense Polynucleotides

The term “antisense polynucleotide” shall be taken to mean a DNA or RNA,or combination thereof, molecule that is complementary to at least aportion of a specific mRNA molecule encoding a FatB or Fad2 polypeptideand capable of interfering with a post-transcriptional event such asmRNA translation. The use of antisense methods is well known in the art(see for example, G. Hartmann and S. Endres, Manual of AntisenseMethodology, Kluwer (1999)). The use of antisense techniques in plantshas been reviewed by Bourque, 1995 and Senior, 1998. Bourque, 1995 listsa large number of examples of how antisense sequences have been utilizedin plant systems as a method of gene inactivation. She also states thatattaining 100% inhibition of any enzyme activity may not be necessary aspartial inhibition will more than likely result in measurable change inthe system. Senior (1998) states that antisense methods are now a verywell established technique for manipulating gene expression.

An antisense polynucleotide of the invention will hybridize to a targetpolynucleotide under physiological conditions. As used herein, the term“an antisense polynucleotide which hybridises under physiologicalconditions” means that the polynucleotide (which is fully or partiallysingle stranded) is at least capable of forming a double strandedpolynucleotide with mRNA encoding a protein, such as those provided inFIG. 2 or 7 or SEQ ID NOs 1 to 4 or 15 to 18 under normal conditions ina cell, preferably a rice cell.

Antisense molecules may include sequences that correspond to thestructural genes or for sequences that effect control over the geneexpression or splicing event. For example, the antisense sequence maycorrespond to the targeted coding region of the genes of the invention,or the 5′-untranslated region (UTR) or the 3′-UTR or combination ofthese. It may be complementary in part to intron sequences, which may bespliced out during or after transcription, preferably only to exonsequences of the target gene. In view of the generally greaterdivergence of the UTRs, targeting these regions provides greaterspecificity of gene inhibition.

The length of the antisense sequence should be at least 19 contiguousnucleotides, preferably at least 50 nucleotides, and more preferably atleast 100, 200, 500 or 1000 nucleotides. The full-length sequencecomplementary to the entire gene transcript may be used. The length ismost preferably 100-2000 nucleotides. The degree of identity of theantisense sequence to the targeted transcript should be at least 90% andmore preferably 95-100%. The antisense RNA molecule may of coursecomprise unrelated sequences which may function to stabilize themolecule.

Catalytic Polynucleotides

The term catalytic polynucleotide/nucleic acid refers to a DNA moleculeor DNA-containing molecule (also known in the art as a “deoxyribozyme”)or an RNA or RNA-containing molecule (also known as a “ribozyme”) whichspecifically recognizes a distinct substrate and catalyzes the chemicalmodification of this substrate. The nucleic acid bases in the catalyticnucleic acid can be bases A, C, G, T (and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence forspecific recognition of a target nucleic acid, and a nucleic acidcleaving enzymatic activity (also referred to herein as the “catalyticdomain”). The types of ribozymes that are particularly useful in thisinvention are the hammerhead ribozyme (Haseloff and Gerlach, 1988;Perriman et al., 1992) and the hairpin ribozyme (Shippy at al., 1999).

The ribozymes of this invention and DNA encoding the ribozymes can bechemically synthesized using methods well known in the art. Theribozymes can also be prepared from a DNA molecule (that upontranscription, yields an RNA molecule) operably linked to an RNApolymerase promoter, e.g., the promoter for 17 RNA polymerase or SP6 RNApolymerase. Accordingly, also provided by this invention is a nucleicacid molecule, i.e., DNA or cDNA, coding for a catalytic polynucleotideof the invention. When the vector also contains an RNA polymerasepromoter operably linked to the DNA molecule, the ribozyme can beproduced in vitro upon incubation with RNA polymerase and nucleotides.In a separate embodiment, the DNA can be inserted into an expressioncassette or transcription cassette. After synthesis, the RNA moleculecan be modified by ligation to a DNA molecule having the ability tostabilize the ribozyme and make it resistant to RNase.

As with antisense polynucleotides described herein, catalyticpolynucleotides of the invention should also be capable of hybridizing atarget nucleic acid molecule (for example an mRNA encoding anypolypeptide provided in FIG. 2 or 7 or SEQ ID NOs 1 to 4 or 15 to 18)under “physiological conditions”, namely those conditions within a cell(especially conditions in a plant cell such as a rice cell).

RNA Interference

RNA interference (RNAi) is particularly useful for specificallyinhibiting the production of a particular protein. Although not wishingto be limited by theory. Waterhouse et al. (1998) have provided a modelfor the mechanism by which dsRNA (duplex RNA) can be used to reduceprotein production. This technology relies on the presence of dsRNAmolecules that contain a sequence that is essentially identical to themRNA of the gene of interest or part thereof in this case an mRNAencoding a polypeptide according to the invention. Conveniently, thedsRNA can be produced from a single promoter in a recombinant vector orhost cell, where the sense and antisense sequences are linked by anunrelated sequence which enables the sense and antisense sequences tohybridize to form the dsRNA molecule with the unrelated sequence forminga loop structure. The design and production of suitable dsRNA moleculesfor the present invention is well within the capacity of a personskilled in the art, particularly considering Waterhouse at al. (1998),Smith at al. (2000), WO 99/32619, WO 99/53050, WO 99/49029, and WO01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology to the targetgene to be inactivated. The DNA therefore comprises both sense andantisense sequences that, when transcribed into RNA, can hybridize toform the double-stranded RNA region. In a preferred embodiment, thesense and antisense sequences are separated by a spacer region thatcomprises an intron which, when transcribed into RNA, is spliced out.This arrangement has been shown to result in a higher efficiency of genesilencing. The double-stranded region may comprise one or two RNAmolecules, transcribed from either one DNA region or two. The presenceof the double stranded molecule is thought to trigger a response from anendogenous plant system that destroys both the double stranded RNA andalso the homologous RNA transcript from the target plant gene,efficiently reducing or eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridise shouldeach be at least 19 contiguous nucleotides, preferably at least 30 or 50nucleotides, and more preferably at least 100, 200, 500 or 1000nucleotides. The full-length sequence corresponding to the entire genetranscript may be used. The lengths are most preferably 100-2000nucleotides. The degree of identity of the sense and antisense sequencesto the targeted transcript should be at least 85%, preferably at least90% and more preferably 95-100%. The RNA molecule may of course compriseunrelated sequences which may function to stabilize the molecule. TheRNA molecule may be expressed under the control of a RNA polymerase IIor RNA polymerase III promoter. Examples of the latter include tRNA orsnRNA promoters.

Preferred small interfering RNA (‘siRNA’) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. Preferably, the target mRNA sequencecommences with the dinucleotide AA, comprises a GC-content of about30-70% (preferably, 30-60%, more preferably 40-60% and more preferablyabout 45%-55%), and does not have a high percentage identity to anynucleotide sequence other than the target in the genome of the plant(preferably rice) in which it is to be introduced, e.g., as determinedby standard BLAST search.

Examples of dsRNA molecules of the invention are provided in Example 5.Further examples include those which comprise a sequence as provided inone or more of SEQ ID NOs 64 to 73 (for Fad2) and SEQ ID NOs 74 to 83(for FatB).

microRNA

MicroRNA regulation is a clearly specialized branch of the RNA silencingpathway that evolved towards gene regulation, diverging fromconventional RNAi/PTGS. MicroRNAs are a specific class of small RNAsthat are encoded in gene-like elements organized in a characteristicinverted repeat. When transcribed, microRNA genes give rise tostem-looped precursor RNAs from which the microRNAs are subsequentlyprocessed. MicroRNAs are typically about 21 nucleotides in length. Thereleased miRNAs are incorporated into RISC-like complexes containing aparticular subset of Argonaute proteins that exert sequence-specificgene repression (see, for example, Millar and Waterhouse, 2005;Pasquinelli et al., 2005; Almeida and Allshire, 2005).

Cosuppression

Another molecular biological approach that may be used isco-suppression. The mechanism of co-suppression is not well understoodbut is thought to involve post-transcriptional gene silencing (PTGS) andin that regard may be very similar to many examples of antisensesuppression. It involves introducing an extra copy of a gene or afragment thereof into a plant in the sense orientation with respect to apromoter for its expression. The size of the sense fragment, itscorrespondence to target gene regions, and its degree of sequenceidentity to the target gene are as for the antisense sequences describedabove. In some instances the additional copy of the gene sequenceinterferes with the expression of the target plant gene. Reference ismade to WO 9720936 and EP 0465572 for methods of implementingco-suppression approaches.

Nucleic Acid Hybridization

In an embodiment, polynucleotides of the invention, or a strand thereof,hybridize under physiological conditions to a polynucleotide comprisingany one or more of the sequence of nucleotides provided as SEQ ID NOs 5to 8, 11 to 14 or 19 to 25. In a further embodiment, polynucleotides ofthe invention, or a strand thereof, also hybridize to a polynucleotidecomprising any one or more of the sequence of nucleotides provided asSEQ ID NOs 5 to 8, 11 to 14 or 19 to 25 under stringent conditions.

As used herein, the phrase “stringent conditions” refers to conditionsunder which a polynucleotide, probe, primer and/or oligonucleotide willhybridize to its target sequence(s), but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures than shorter sequences. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength, pH and nucleicacid concentration) at which 50% of the probes complementary to thetarget sequence hybridize to the target sequence at equilibrium. Sincethe target sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

Stringent conditions are known to those skilled in the art and can befound in Ausubel et al (supra), Current Protocols In Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, as well as the Examplesdescribed herein. Preferably, the conditions are such that sequences atleast about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to eachother typically remain hybridized to each other. A non-limiting exampleof stringent hybridization conditions are hybridization in a high saltbuffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C., followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. Inanother embodiment, a nucleic acid sequence that is hybridizable to thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs 5to 8, 11 to 14 or 19 to 25, under conditions of moderate stringency isprovided. A non-limiting example of moderate stringency hybridizationconditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDSand 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one ormore washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderatestringency that may be used are well-known within the art, see, e.g.,Ausubel at al. (supra), and Kriegler, 1990; Gene Transfer AndExpression, A Laboratory Manual, Stockton Press, NY. In yet anotherembodiment, a nucleic acid that is hybridizable to the nucleic acidmolecule comprising the nucleotide sequences SEQ ID NOs 5 to 8, 11 to 14or 19 to 25, under conditions of low stringency, is provided. Anon-limiting example of low stringency hybridization conditions arehybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmonsperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one ormore washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDSat 50° C. Other conditions of low stringency that may be used are wellknown in the art, see, e.g., Ausubel et al. (supra) and Kriegler, 1990,Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY,as well as the Examples provided herein.

Nucleic Acid Constructs, Vectors and Host Cells

The present invention includes the production of genetically modifiedrice plants, wherein the plant has decreased expression of a polypeptidehaving Fad2 and/or FatB activity relative to a correspondingnon-modified plant.

Nucleic acid constructs useful for producing the above-mentionedtransgenic plants can readily be produced using standard techniques.

When inserting a region encoding an mRNA the construct may compriseintron sequences. These intron sequences may aid expression of thetransgene in the plant. The term “intron” is used in its normal sense asmeaning a genetic segment that is transcribed but does not encodeprotein and which is spliced out of an RNA before translation. Intronsmay be incorporated in a 5′-UTR or a coding region if the transgeneencodes a translated product, or anywhere in the transcribed region ifit does not. However, in a preferred embodiment, any polypeptideencoding region is provided as a single open reading frame. As theskilled addressee would be aware, such open reading frames can beobtained by reverse transcribing mRNA encoding the polypeptide.

To ensure appropriate expression of the gene encoding an mRNA ofinterest, the nucleic acid construct typically comprises one or moreregulatory elements such as promoters, enhancers, as well astranscription termination or polyadenylation sequences. Such elementsare well known in the art.

The transcriptional initiation region comprising the regulatoryelement(s) may provide for regulated or constitutive expression in theplant. Preferably, expression at least occurs in cells of the embryo,endosperm, bran layer developing seed and/or mature seed (grain). In analternate embodiment, the regulatory elements may be promoters notspecific for seed cells (such as ubiquitin promoter or CaMV35S orenhanced 35S promoters).

Examples of seed specific promoters useful for the present inventioninclude, but are not limited to, the wheat low molecular weight gluteninpromoter (Colot at al., 1987), the promoter expressing α-amylase inwheat seeds (Stefanov at al., 1991), and the hordein promoter (Brandt etal., 1985).

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter. Thesepromoters have been used to create DNA vectors that have been expressedin plants; see, e.g., PCT publication WO 8402913. All of these promotershave been used to create various types of plant-expressible recombinantDNA vectors.

The promoter may be modulated by factors such as temperature, light orstress. Ordinarily, the regulatory elements will be provided 5′ of thegenetic sequence to be expressed. The construct may also contain otherelements that enhance transcription such as the nos 3′ or the ocs 3′polyadenylation regions or transcription terminators.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the polynucleotideof the present invention, and can be specifically modified if desired soas to increase translation of mRNA. For a review of optimizingexpression of transgenes, see Koziel at al. (1996). The 5 non-translatedregions can also be obtained from plant viral RNAs (Tobacco mosaicvirus, Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaicvirus, among others) from suitable eukaryotic genes, plant genes (wheatand maize chlorophyll a/b binding protein gene leader), or from asynthetic gene sequence. The present invention not limited to constructswherein the non-translated region is derived from the 5′ non-translatedsequence that accompanies the promoter sequence. The leader sequencecould also be derived from an unrelated promoter or coding sequence.Leader sequences useful in context of the present invention comprise themaize Hsp70 leader (U.S. Pat. No. 5,362,865 and U.S. Pat. No.5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the chimeric vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ translated region, the 3′ untranslated region from pea small subunitRubisco gene, the 3′ untranslated region from soybean 75 seed storageprotein gene are commonly used in this capacity. The 3′ transcribed,non-translated regions containing the polyadenylate signal ofAgrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

Typically, the nucleic acid construct comprises a selectable marker.Selectable markers aid in the identification and screening of plants orcells that have been transformed with the exogenous nucleic acidmolecule. The selectable marker gene may provide antibiotic or herbicideresistance to the rice cells, or allow the utilization of substratessuch as mannose. The selectable marker preferably confers hygromycinresistance to the rice cells.

Preferably, the nucleic acid construct is stably incorporated into thegenome of the plant. Accordingly, the nucleic acid comprises appropriateelements which allow the molecule to be incorporated into the genome, orthe construct is placed in an appropriate vector which can beincorporated into a chromosome of a plant cell.

One embodiment of the present invention includes a recombinant vector,which includes at least one polynucleotide molecule of the presentinvention, inserted into any vector capable of delivering the nucleicacid molecule into a host cell. Such a vector contains heterologousnucleic acid sequences, that is nucleic acid sequences that are notnaturally found adjacent to nucleic acid molecules of the presentinvention and that preferably are derived from a species other than thespecies from which the nucleic acid molecule(s) are derived. The vectorcan be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al. Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al, Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred host cells are plant cells, morepreferably cells of a cereal plant, and even more preferably a ricecell.

Transgenic Plants

Transgenic rice (also referred to herein as genetically modified rice)can be produced using techniques known in the art, such as thosegenerally described in A. Slater et al., Plant Biotechnology—The GeneticManipulation of Plants, Oxford University Press (2003), and P. Christouand H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons(2004).

In a preferred embodiment, the transgenic plants are homozygous for eachand every polynucleotide that has been introduced (transgene) so thattheir progeny do not segregate for the desired phenotype. The transgenicplants may also be heterozygous for the introduced transgene(s), suchas, for example, in F1 progeny which have been grown from hybrid seed.Such plants may provide advantages such as hybrid vigour, well known inthe art.

Four general methods for direct delivery of a gene into cells have beendescribed: (1) chemical methods (Graham et al., 1973); (2) physicalmethods such as microinjection (Capecchi, 1980); electroporation (see,for example, WO 87/06614, U.S. Pat. No. 5,472,869, 5,384,253, WO92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat.No. 4,945,050 and U.S. Pat. No. 5,141,131); (3) viral vectors (Clapp,1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediatedmechanisms (Curiel at al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. An illustrative embodiment of a method for delivering DNA intoZea mays cells by acceleration is a biolistics α-particle deliverysystem, that can be used to propel particles coated with DNA through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with corn cells cultured in suspension. A particle deliverysystem suitable for use with the present invention is the heliumacceleration PDS-1000/He gun is available from Bio-Rad Laboratories.

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain up to 1000 or more foci ofcells transiently expressing a marker gene. The number of cells in afocus that express the exogenous gene product 48 hours post-bombardmentoften range from one to ten and average one to three.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Method disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S.Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265.

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions that influence the physiological state of therecipient cells and that may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,104,310, U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135).Further, the integration of the T-DNA is a relatively precise processresulting in few rearrangements. The region of DNA to be transferred isdefined by the border sequences, and intervening DNA is usually insertedinto the plant genome.

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyarna et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized stages of embryonicdevelopment through the rooted plantlet stage. Transgenic embryos andseeds are similarly regenerated. The resulting transgenic rooted shootsare thereafter planted in an appropriate plant growth medium such assoil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

Methods for transformation of cereal plants such as rice for introducinggenetic variation into the plant by introduction of an exogenous nucleicacid and for regeneration of plants from protoplasts or immature plantembryos are well known in the art, see for example, Canadian PatentApplication No. 2,092,588, Australian Patent Application No 61781/94,Australian Patent No 667939, U.S. Pat. No. 6,100,447, InternationalPatent Application PCT/US97/10621, U.S. Pat. No. 5,589,617, U.S. Pat.No. 6,541,257, and other methods are set out in Patent specificationWO99/14314. Preferably, transgenic rice plants are produced byAgrobacterium tumefaciens mediated transformation procedures. An exampleof Agrobacterium mediated transformation of rice is provided herein inExample 5. Vectors carrying the desired nucleic acid construct may beintroduced into regenerable rice cells of tissue cultured plants orexplants, or suitable plant systems such as protoplasts.

The regenerable rice cells are preferably from the scutellum of immatureembryos, mature embryos, callus derived from these, or the meristematictissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

Marker Assisted Selection

Marker assisted selection is a well recognised method of selecting forheterozygous plants required when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants. To further speed up the backcrossingprogram, the embryo from immature seeds (25 days post anthesis) may beexcised and grown up on nutrient media under sterile conditions, ratherthan allowing full seed maturity. This process, termed “embryo rescue”,used in combination with DNA extraction at the three leaf stage andanalysis for the desired genotype allows rapid selection of plantscarrying the desired trait, which may be nurtured to maturity in thegreenhouse or field for subsequent further backcrossing to the recurrentparent.

Any molecular biological technique known in the art which is capable ofdetecting a Fad2 or FatB gene can be used in the methods of the presentinvention. Such methods include, but are not limited to, the use ofnucleic acid amplification, nucleic acid sequencing, nucleic acidhybridization with suitably labeled probes, single-strand conformationalanalysis (SSCA), denaturing gradient gel electrophoresis (DOGE),heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalyticnucleic acid cleavage or a combination thereof (see, for example,Lemieux, 2000; Langridge et al., 2001). The invention also includes theuse of molecular marker techniques to detect polymorphisms linked toalleles of (for example) a Fad2 or FatB gene which confer the desiredphenotype. Such methods include the detection or analysis of restrictionfragment length polymorphisms (RFLP), RAPD, amplified fragment lengthpolymorphisms (AFLP) and microsatellite (simple sequence repeat, SSR)polymorphisms. The closely linked markers can be obtained readily bymethods well known in the art, such as Bulked Segregant Analysis, asreviewed by Langridge et al. (2001).

The “polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are known in theart, and are taught, for example, in “PCR” (Ed. M. J. McPherson and S. GMoller (2000) BIOS Scientific Publishers Ltd, Oxford). PCR can beperformed on cDNA obtained from reverse transcribing mRNA isolated fromplant cells. However, it will generally be easier if PCR is performed ongenomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridisingin a sequence specific fashion to the target sequence and being extendedduring the PCR. Amplicons or PCR products or PCR fragments oramplification products are extension products that comprise the primerand the newly synthesized copies of the target sequences. Multiplex PCRsystems contain multiple sets of primers that result in simultaneousproduction of more than one amplicon. Primers may be perfectly matchedto the target sequence or they may contain internal mismatched basesthat can result in the introduction of restriction enzyme or catalyticnucleic acid recognition/cleavage sites in specific target sequences.Primers may also contain additional sequences and/or contain modified orlabelled nucleotides to facilitate capture or detection of amplicons.Repeated cycles of heat denaturation of the DNA, annealing of primers totheir complementary sequences and extension of the annealed primers withpolymerase result in exponential amplification of the target sequence.The terms target or target sequence or template refer to nucleic acidsequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.(supra) and Sambrook et at (supra). Sequencing can be carried out by anysuitable method, for example, dideoxy sequencing, chemical sequencing orvariations thereof. Direct sequencing has the advantage of determiningvariation in any base pair of a particular sequence.

Hybridization based detection systems include, but are not limited to,the TaqMan assay and molecular beacons. The TaqMan assay (U.S. Pat. No.5,962,233) uses allele specific (ASO) probes with a donor dye on one endand an acceptor dye on the other end such that the dye pair interact viafluorescence resonance energy transfer (FRET). A target sequence isamplified by PCR modified to include the addition of the labeled ASOprobe. The PCR conditions are adjusted so that a single nucleotidedifference will effect binding of the probe. Due to the 5 nucleaseactivity of the Taq polymerase enzyme, a perfectly complementary probeis cleaved during PCR while a probe with a single mismatched base is notcleaved. Cleavage of the probe dissociates the donor dye from thequenching acceptor dye, greatly increasing the donor fluorescence.

An alternative to the TaqMan assay is the molecular beacon assay (U.S.Pat. No. 5,925,517). In the molecular beacon assay, the ASO probescontain complementary sequences flanking the target specific species sothat a hairpin structure is formed. The loop of the hairpin iscomplimentary to the target sequence while each arm of the hairpincontains either donor or acceptor dyes. When not hybridized to a donorsequence, the hairpin structure brings the donor and acceptor dye closetogether thereby extinguishing the donor fluorescence. When hybridizedto the specific target sequence, however, the donor and acceptor dyesare separated with an increase in fluorescence of up to 900 fold.Molecular beacons can be used in conjunction with amplification of thetarget sequence by PCR and provide a method for real time detection ofthe presence of target sequences or can be used after amplification.

Tilling

Plants of the invention can be produced using the process known asTILLING (Targeting Induced Local Lesions IN Genomes). In a first step,introduced mutations such as novel single base pair changes are inducedin a population of plants by treating seeds (or pollen) with a chemicalmutagen, and then advancing plants to a generation where mutations willbe stably inherited. DNA is extracted, and seeds are stored from allmembers of the population to create a resource that can be accessedrepeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cel I, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TELLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique.

TILLING is further described in Slade and Knauf (2005) and Henikoff etal. (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Mutagenesis Procedures

Techniques for generating mutant rice plant lines are known in the art.Examples of mutagens that can be used for generating mutant rice plantsinclude irradiation and chemical mutagenesis. Mutants may also beproduced by techniques such as T-DNA insertion and transposon-inducedmutagenesis. The mutagenesis procedure may be performed on any parentalcell of a rice plant, for example a seed or a parental cell in tissueculture.

Chemical mutagens are classifiable by chemical properties, e.g.,alkylating agents, cross-linking agents, etc. Useful chemical mutagensinclude, but are not limited to, N-ethyl-N-nitrosourea (ENU);N-methyl-N-nitrosourea (MNU); procarbazine hydrochloride; chlorambucil;cyclophosphamide; methyl methanesulfonate (MMS); ethyl methanesulfonate(EMS); diethyl sulfate; acrylamide monomer, triethylene melamine (TEM);melphalan; nitrogen mustard; vincristine; dimethylnitrosamine;N-methyl-N′-nitro-Nitrosoguani-dine (MNNG); 7,12 dimethylbenzanthracene(DMBA); ethylene oxide; hexamethylphosphoramide; and bisulfan.

An example of suitable irradiation to induce mutations is by gammaradiation, such as that supplied by a Cesium 137 source. The gammaradiation preferably is supplied to the plant cells in a dosage ofapproximately 60 to 200 Krad., and most preferably in a dosage ofapproximately 60 to 90 Krad.

Plants are typically exposed to a mutagen for a sufficient duration toaccomplish the desired genetic modification but insufficient tocompletely destroy the viability of the cells and their ability to beregenerated into a plant.

Antibodies

Monoclonal or polyclonal antibodies which bind specifically to FatB orFad2 polypeptides can be useful for some of the methods of theinvention.

The term “binds specifically” refers to the ability of the antibody tobind to a FatB or Fad2 polypeptide but not other proteins of rice,especially proteins of rice seeds.

As used herein, the term “epitope” refers to a region of a FatB or Fad2polypeptide which is bound by the antibody. An epitope can beadministered to an animal to generate antibodies against the epitope,however, antibodies useful for the methods described herein preferablyspecifically bind the epitope region in the context of the entirepolypeptide.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptidesuch as those provided in FIG. 2 or 7 or SEQ NOs 1 to 4 or 15 to 18.Serum from the immunised animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies containsantibodies to other antigens, the polyclonal antibodies can be purifiedby immunoaffinity chromatography. Techniques for producing andprocessing polyclonal antisera are known in the art. In order that suchantibodies may be made, the invention also provides peptides of theinvention or fragments thereof haptenised to another peptide for use asimmunogens in animals.

Monoclonal antibodies directed against polypeptides of the invention canalso be readily produced by one skilled in the art. The generalmethodology for making monoclonal antibodies by hybridomas is wellknown. Immortal antibody-producing cell lines can be created by cellfusion, and also by other techniques such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.Panels of monoclonal antibodies produced can be screened for variousproperties; i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. Such fragmentsinclude Fv, F(ab′) and F(ab′)₂ fragments, as well as single chainantibodies (scFv). Furthermore, the antibodies and fragments thereof maybe humanised antibodies, for example as described in EP-A-239400.

Antibodies may be bound to a solid support and/or packaged into kits ina suitable container along with suitable reagents, controls,instructions and the like.

Preferably, the antibodies are delectably labeled. Exemplary detectablelabels that allow for direct measurement of antibody binding includeradiolabels, fluorophores, dyes, magnetic beads, chemiluminescers,colloidal particles, and the like. Examples of labels which permitindirect measurement of binding include enzymes where the substrate mayprovide for a coloured or fluorescent product. Additional exemplarydetectable labels include covalently bound enzymes capable of providinga detectable product signal after addition of suitable substrate.Examples of suitable enzymes for use in conjugates include horseradishperoxidase, alkaline phosphatase, malate dehydrogenase and the like.Where not commercially available, such antibody-enzyme conjugates arereadily produced by techniques known to those skilled in the art.Further exemplary detectable labels include biotin, which binds withhigh affinity to avidin or streptavidin; fluorochromes (e.g.,phycobiliproteins, phycoerythrin and allophycocyanins; fluorescein andTexas red), which can be used with a fluorescence activated cell sorter;haptens; and the like. Preferably, the detectable label allows fordirect measurement in a plate luminometer, e.g., biotin. Such labeledantibodies can be used in techniques known in the art to detectpolypeptides of the invention.

EXAMPLES Example 1 Materials and Methods

Extraction of Oil with Sodium Methoxide

For fatty acid and other analyses, total lipid was extracted from ricegrains as follows unless stated otherwise. In some cases, samples eachconsisting of a half grain were used for the extraction, with the secondhalf-grain containing the embryo being used for embryo rescue. Thetechnique can also be used with other cereals.

A single developing seed or half-seed was squashed between filter papersand placed in a tube. 2 ml 0.5M sodium methoxide was added, the tubesealed tightly and then incubated at 80° C. for 10 min. After the tubewas cooled, 0.1 ml glacial acetic acid was added followed by 2 ml ofdistilled water and 2 ml of petroleum spirit. The mixtures were vortexedfor 10 sec and, after the phases had separated, the upper petroleumlayer was transferred to a small test tube. Approximately 1g ofpotassium bicarbonate/sodium sulphate mixture was added to the testtubes and the mixture vortexed. The sample solutions were transferred toautosampler vials and stored in a freezer at −20° C. until GC analysiswas performed as described by Soutjesdic at al. (2002).

Extraction of Lipids from Tissues with High Water Content (Bligh-DyerMethod)

This method was adapted from Bligh and Dyer (1959). 1.5 ml of CHCl₃/MeOH(1:2) was added to a tissue sample in 0.4 ml of buffer and the samplevortexed vigorously. A further 0.5 ml of CHCl₃ was added and the samplevortexed again. 0.5 ml of H₂O was added and the sample vortexed again.The tube was centrifuged briefly at 3000 rpm to separate phases; a whiteprecipitate appeared at the interface. The organic (lower) phase wastransferred to a new tube and concentrated under vacuum. If acidiclipids were to be extracted, 0.5 ml of 1% HClO₄ was added instead of 0.5ml of H₂O. The volumes in the above procedure could be modified as longas the ratios of CHCl₃/MeOH/H₂O were maintained.

Preparation of Fatty Acid Methyl Esters (FAME) for QuantitativeDetermination of Fatty Acid Content

To prepare FAME directly from grain, 10-15 seed were weighed accuratelyand transferred to a glass tube. An internal standard of 10 μl of 1mg/ml 17:0-methyl ester was added to each seed sample. 0.75 ml 1 Nmethanolic-HCl (Supelco) was added to each tube which was capped tightlyand refluxed at 80° C. for at least 2-3 hrs or overnight Samples werecooled, 0.5 ml NaCl (0.9% w/v) added followed by 300 μl hexane. Thetubes were capped again and vortexed vigorously. The upper hexane phase(200-250 μl) was carefully transferred to an eppendorf tube. The samplewas dried down completely under nitrogen. The dried FAME samples weredissolved in 20 μl of hexane and transferred to conical glass inserts invials for GC analysis.

FA Analysis by Gas Chromatography

Fatty acid methyl esters were prepared by alkaline transmethylation asfollows. Single seed samples were squashed between filter paper disks.The fatty acids in the lipid transferred to the filter paper disks werethen methylated in 2 mL of 0.02 M sodium methoxide for 10 minutes at 80°C., followed by cooling for 30 minutes. 0.1 mL of glacial acetic acidwas then added, followed in order by 2 mL of distilled water and 2 mL ofhexane. After vortexing and phase separation, the upper hexane layercontaining the fatty acid methyl esters was transferred to a microvial.Fatty acid methyl esters were analysed by gas-liquid chromatography aspreviously described (Stoutjesdijk et al., 2002).

Transformation of Rice

Rice (cv. Nipponbare) was transformed as follows.

i) Induction and Culture

Husks were removed from mature grain, which were then soaked in 70% EtOHfor 30 sec to remove any outer layer of wax. The cleaned grains werewashed 3 times with sterile H₂0 and soaked in a solution of 25% bleach(with 2 drops Tween-20 detergent added) for 20 minutes with shaking tosurface sterilize them. Under asceptic conditions, the grains wererinsed briefly with 70% EtOH, washed thoroughly with sterile H₂0 8-10times and plated onto N₆D medium. Plates were sealed with Micropore-tapeand incubated under full light at 28° C. Calli were produced after 6-8weeks and then transferred to NB media. They were sealed with paraffinpaper and left in the at 28° C. with sub-culturing every 4 weeks ontofresh NB plates. Calli were not used for transformation after more then5 subcultures.

ii) Transformation

Healthy looking calli were picked from subculture plates and transferredonto fresh NB plates at a density of 25-30 calli/plate. Two days later,a fresh culture was established of the Agrobacterium strain containingthe construct to be used, and incubated at 28° C. The culture medium wasNB medium supplemented with 100 μM acetosyringone. Calli were immersedin a suspension of the cells for 10 minutes. After draining off excesssuspension, calli were placed on NB medium supplemented with 100 μMacetosyringone and incubated in the dark at 25° C. for 3 days(co-cultivation). After the co-cultivation step, calli were washedgently three times in a tube with sterile H₂0 containing 150 mg/mlTimetin. Calli were blotted dry on filter paper and plated well spacedonto NBCT plates (containing 100 μg/ml kanamycin if a kanamycinselectable marker gene was used or other selection agent as appropriate,150 μg/ml Timentin and 200 μg/ml Claroforan). The plates were incubatedin the dark for 3-4 weeks at 26-28° C. Resistant calli were observedafter about 10 days and transferred to NBCT+selection plates andincubated in the dark for a further 14-21 days. Healthy call weretransferred onto PRCT+selection plates and incubated in the dark for8-12 days, then transferred onto RCT+selection plates and incubated infull light at 28° C. for 30 days. After this time, plantlets that haddeveloped were transferred to ½MS medium in tissue culture pots andincubated under light for 10-14 days for further growth before transferto soil.

iii) Media Composition and Components for Rice Tissue Culture

N6 macro-elements (20×) (g/l): (NH₄)₂SO₄, 9.3; KNO₃, 56.6; KH₂PO₄, 8;MgSO₄.7H₂O, 3.7; CaCl₂.2H₂O, 3.3.

N6 micro (1000×) (mg/100 ml): MnSO₄.4H₂O, 440; ZnSO₄.7H₂O, 150; H₃BO₃,160; KI, 80.

N6 Vitamins (100×) (mg/100 ml): Glycine, 20; Thiamine-HCl, 10;Pyridoxine-HCl, 5; Nicotinic acid, 5.

B5 micro-elements (100×) (mg/1000 ml): MnSO₄.4H₃O, 1000; Na₂MoO₄.2H₂O,25; H₃BO₃, 300; ZnSO₄.7H₃O, 200; CuSO₄.5H₂O, 3.87; CoCl₂.6H₂O, 2.5; KI,75.

B5 vitamins (100×) and Gamborg's vitamin solution (1000×) from Sigmacell culture.

FeEDTA (200×) (g/200 ml): Ferric-sodium salt, 1.47.2,4-D (1 mg/ml): Dissolve 100 mg of 2,4-dichloro-phenoxyacetic acid in 1ml absolute ethanol, add 3 ml of 1N KOH, adjust to pH 6 with 1N HCl.

BAP (1 mg/ml): 6-benzyl amino purine from Sigma cell culture.

NAA (1 mg/ml): Naphthalene acetic acid from Sigma cell culture.

ABA (2.5 mg/ml): Dissolve 250 mg of Abscisic acid in 2 ml of 1M NaOH,make up to 100 ml with sterile distilled water. Final backgroundconcentration to be 20 mM NaOH.

Hygromycin (50 mg/ml): Hygromycin solution from Roche.

Timetin (150 mg/ml): Dissolve 3100 mg of Timetin in 20.66 ml of sterilewater. Final concentration to be 150 mg/ml.

Claforan (200 mg/ml): Dissolve 4 gm of Claforan in 20 ml of sterilewater.

MS salts: Murashige-Skoog minimal organic medium.

N6D medium (amount per litre):

N6 macro (10X) 100 ml N6 micro (1000X) 1 ml N6 vitamins (1000X) 1 ml MSiron/EDTA 5 ml Myoinositol 100 mg Casamino acid 300 mg Proline 2.9 g2,4-D (1 mg/ml) 2 ml Sucrose 30 g

pH adjusted to 5.8 with 1M KOH, add 3g phytogel/litre, autoclave.

NB medium (amount per litre):

N6 macro-elements (20X) 50 ml B5 micro-elements (100) 10 ml B5 vitamins(100X) 10 ml FeEDTA (200X) 5 ml 2,4-D (1 mg/ml) 2 ml Sucrose 30 gProline 500 mg Glutamine 500 mg Casein enzymatic hydrolysate (CEH) 300mg

NBO: NB media, plus 30 gll mannitol and 30g/l sorbitol, added before pHadjustment.

NBCT+selection (Hygromycin-H30): NB media plus 30 mg/l hygromycin,Timentin 1S0 mg/ml, Claforan 200 mg/l.

NBCT+selection (Hygromycin-HSO); NB media plus selection 50 mg/lhygromycin, 200 mg/l Claforan and Timentin 150 mg/ml.

PRCT+Selection: NB media with no 2,4-D, plus following added afterautoclave: BAP, 2 mg/l; NAA, 1 mg/l; ABA, 5 mg/l; Claforan, 100g/l;Timetin; 150 mg/ml+selection.

RCT+Selection: NB media with no 2,4-D, plus following added afterautoclave: BAP; 3 mg/l; NAA, 0.5 mg/l; Timetin, 150 mg/ml; Claforan, 100mg/l+selection.

½ MS media: MS salts and vitamin mixture, 2.21 g; Sucrose, 109 perlitre, add 2.5 g phytogel/l. after autoclaving add 0.05 mg/l NAA andTimentin 150 mg/ml.

Example 2 Identification and Isolation of FutB Genes from Rice

FutB genes encode the enzyme palmitoyl-ACP thioesterase which have theactivity preferentially transferring fatty acids that have a length of16 carbons or less from acyl carrier protein to acyl-CoA and thusprevent further elongation of the fatty acid carbon chain. Putative riceFatB sequences were identified using homology based searches using thesequence for the Genbank accession Arabidopsis locus AtACPTE32 and Irislocus AF213480. The program used was Megablast available at NCBI(www.ncbi.nlm.nih.gov/). Default parameters used by NCBI were used andthe databases used were both non-redundant (nr) and high throughput genesequences (htgs) for rice, Oryza sativa. The most similar sequences fromrice selected by the Megablast program were then translated and examinedfor the presence of the conserved sequence NQHVNN (SEQ ID NO:26) foundin all FatB sequences. A further amino acid residue believed to beessential in Arabidopsis is cysteine 264 and along with asparagine 227and histidine 229 (which are both present in the conserved sequenceNQHVNN) comprise the proposed catalytic triad. The Chinese Rice Database(current Website address rise.genomics.org.cn/rice/index2.jsp) was alsoused to a limited extent and default parameters for BLAST searching wereused with the Arabidopsis sequence and Iris sequences. The translatedsequences are aligned in FIG. 2.

An overview of the structures of all the FatB genes is shown as in FIG.3. The genes described correspond as follows to the protein sequencesdiscussed—AC108870 corresponds to Os11g43820, AP005291 corresponds toOs02g43090, AP000399 corresponds to Os06g5130 and AP004236 correspondsto Os06g39520. Note the possibility of multiple transcripts from onegene as indicated on the diagram. FIG. 4 shows a CLUSTAL W (fast)alignment of the ‘gene’ sequences using default parameters-note that the‘genes’ are of different lengths.

The genes comprise 6 exons. The structure of the gene described byOs06g5130 is shown in detail FIG. 5,

FIG. 6 shows an alignment of the coding sequences of the FatB cDNAsindicating the different primers used for selective PCR amplification.

The sequence identity between the translated peptide sequence ofOs06g5130 and Os06g39520 (corresponding to sequence ap000399 andap004236 in FIG. 2) was 74% overall, and the identity at the nucleotidelevel over the entire coding sequence was 69%. In both cases the programBESTFIT with default parameters was used. The polypeptides deduced fromOs02g43090 (one transcript). Os06g05130, Os06g39520 and Os011g43820correspond to 298, 427, 423 and 425 amino acids respectively.

Activity of the encoded proteins was surmised from the high degree ofsequence identity with known polypeptides shown to have this activity.Furthermore, the observed effect on palmitic acid levels of geneinactivating constructs based on these sequences was also consistentwith this conclusion (see below).

Expression of the gene family was complex, with at least seventranscripts predicted from these four genes. On the basis of RT-PCRexperiments that were performed and relative numbers of clones recoveredfrom EST libraries, it appeared that RNA from Os06g5130 was relativelyabundant in the grain, while Os11g43820 was expressed at a moderatelevels in that tissue and the other genes were only expressed at a lowlevel.

Example 3 Identification and Isolation of Fad2 Genes from Rice

Proteins encoded by Fad2 genes (Fatty acid desaturase 2) are responsiblefor the introduction of a double bond into 18:1 fatty acids—they are Δ12desaturases. The Genbank sequence from the Arabiodopsis locus athd12aaawas used to search the or and htgs databases for Oryza sativa usingMegablast at default settings. The most similar sequences retrieved fromrice were translated and examined for the presence of conservedhydrophobic motifs FSYVVHDLVIVAALLFALVMI (SEQ ID NO:27),AWPLYIAQGCVLTGVWVIA (SEQ ID NO:28), ISDVGVSAGLALFKLSSAFGF (SEQ IDNO:29), VVRVYGVPLLIVNAWLVLITYLQ (SEQ ID NO:30) and the histidine ricesequences HECGHH (SEQ ID NO:31), HRRHHA (SEQ ID NO:32) and HVAHH (SEQ IDNO:33). The translated amino acid sequences of the isoforms obtained areshown in FIG. 7.

FIG. 8 provides an alignment of Fad2 sequences showing location of 5′UTR in isoform AP004047 (lowercase) and location of primers used foramplification by RT-PCR (underlined). The location of the stop codon isindicated by a box and untranslated regions downstream of the stop codonare in lower case.

Four gene sequences are recovered as being highly similar from the ricegenome when the nucleotide sequence encoding the protein with the aminoacid sequence of AP004047 was used to search the rice genome using theprogram BLAST with the default parameters. The overall structure ofthese genes are shown in FIG. 9. The genes correspond to the proteinsequences as follows. The protein sequence AP004047 (also called FAD2-1herein) corresponded to the gene Os02g48560, the sequence AP005168 (alsocalled FAD2-2 herein) corresponded to Os07g23410, and the sequencecontig2654 corresponded to Os07g23430. In addition there was a sequencethat shared an intriguing extent of sequence identity but was clearlydifferent to these sequences and may be a pseudo-gene. This sequence wasOs07g23390.

Unlike the FatB genes, the Fad2 genes did not contain any introns. Analignment of all the protein coding sequences is shown in FIG. 10. Thesequence identity over the entire coding region for Os02g48560 toOs07g23410 was 79%.

The molecular weight of the polypeptide encoded by Os02g48560 (ieAP004047) was 44.35 kDa before processing and contained 388 amino acids.The molecular weight of the polypeptide encoded by Os07g23410 was 44.9kDa and contained 390 amino acids. These deduced polypeptides had 77%sequence identity as determined by the program BESTFIT using defaultparameters. The deduced polypeptide from Os07g23430 had a molecularweight of 41 kDa (363 amino acids) and from Os07g23390 was 24 kDa (223amino acids). An alignment of all the deduced polypeptide sequences isshown in FIG. 7.

The sequence corresponding to Os02g48560 was expressed in the grain andthis observation was consistent with data from the relative frequenciesof clones for this gene in EST libraries where cognate sequences wererecovered from grain cDNA libraries. Sequences corresponding to two ofthe isoforms encoded on chromosome 7 (Os07g23430,23410) have beenrecovered from a leaf EST library but the sequence corresponding to theother gene (Os07g23390) has not yet been reported; we concluded it maybe expressed at low levels if at all.

In conclusion, the rice Fad2 gene family was also a complex gene family.The two transcripts deduced from Os02g48560 were indistinguishable bysequence. The sequence deduced from Os02g48560 was clearly expressed inthe grain.

Example 4 Expression of FatB and Fad2 Genes in Rice

To determine which, if any, of the 4 putative FatB and 3 Fad2 genesidentified in rice as described in Examples 2 and 3 might be expressedin developing rice grain, reverse transcription polymerase chainreaction (RT-PCR) assays were carried out. Since the genes were closelyrelated in sequence, primers had to be designed that were specific foreach of the genes, to assay transcripts for each gene specifically.Regions of sequence divergence were identified from the alignments(FIGS. 6 and 8) and several primer pairs were designed and tested.Primer sequences for detecting expression of the putative FatB genes aregiven in Tables 3 and 4. As an internal standard against which tocompare expression levels, RT-PCR was also carried out on the RNA forexpression oldie rice gene encoding alpha-tubulin, OsTubA1. This genewas known to be expressed in all actively dividing tissues and was notaffected by the hormone ABA and so was suitable as a constitutivelyexpressed control for leaf and grain analysis.

RNA was prepared from rice grains approx 15 days after flowering usingthe Qiagen RNeasy kit following protocols supplied by the manufacturer.DNAse treatment (DNA-free kit, Ambion) was then used to removecontaminating DNA from the RNA preparation. The RT-PCR mix contained 5μl of 5× Qiagen OneStep RT-PCR buffer, 1 μl of dNTP mix (containing 10mM of each dNTP), 15 pmol of each primer and approximately 20 pg of RNAto a final volume of 25 μl. The following RT-PCR cycling program wasused for the RT-PCR amplifications: 30 min at 50° C. (reversetranscription), 15 min at 95° C. PCR activation), 30 cycles of (1 min at94° C., 1 min at 57° C., 1 min at 72° C.) (PCR amplification), then afinal extension of 10 min at 72° C.

TABLE 3 Primers designed to amplify and discriminate relative expressionof the putative FatB transcripts using one-step RT-PCR. GenbankID/ Chinese Gene Contig no. Amplified ID Primer name Primer SequenceFatB-1 AP000399 p0399F2 CGCTGCTACCAAACAATTCA (SEQ ID NO: 34) p0399R2TTCTGTGTTGCCATCATCG (SEQ ID NO: 35) FatB-2 AC108870 p5291_F2CAGGAAATAAAGTTGGTGATGATG (SEQ ID NO: 36) p8870R CTTCACAATATCAGCTCCTGACTC(SEQ ID NO: 37) FatB-3 AP005291 p5291_F2 CAGGAAATAAAGTTGGTGATGATG(SEQ ID NO: 38) p5291R CTTCACAATGTCAGCCTTCAC (SEQ ID NO: 39) FatB-4AP004236 p4236F2 ACAGGCCTGACTCCACGAT (SEQ ID NO: 40) p4236R2GTCCAGAGTGCTTGTTGCAG (SEQ ID NO: 41) OsTubA1 AF182523 OSTUBA1_FTACCCACTCCCTCCTTGAGC (SEQ ID NO: 42) OSTUBA1_R AGGCACTGTTGGTGATCTCG(SEQ ID NO: 43)

TABLE 4 Primers designed to amplify and discriminate relative expressionof the putative Fad2 transcripts using one-step RT-PCR. Genbank ID/ GeneChinese Amplified Contig no. ID Primer name Primer Sequence Fad2-1AP004047 pFad2-1F CACAAAGAGGGAGGGAACAA (SEQ ID NO: 44) pFad2-1RGAAGGACTTGATCACCGAGC (SEQ ID NO: 45) Fad2-2 Contig2654 UTR_2654_FCACAACATCACGGACACACA (SEQ ID NO: 46) UTR_2654_R GCAAGACCGACATGGCTAAT(SEQ ID NO: 47) Fad2-3 AP005168 UTR_5168_F ACGTCCTCCACCACCTCTT(SEQ ID NO: 48) UTR_5168_R CAGAAGCAGTGACATACCCAAG (SEQ ID NO: 49)OsTubA1 AF182523 OSTUBA1_F TACCCACTCCCTCCTTGAGC (SEQ ID NO: 50)OSTUBA1_R AGGCACTGTTGGTGATCTCG (SEQ ID NO: 51)

Results from the RT-PCR experiments demonstrated that the sequenceFad2-1 gene (TIGR rice database identifier LOC_Os02g48560) was highlyexpressed in both grain and leaf relative to the genes encoding theother isoforms. The other two Fad2 genes (LOC_Os07g23410 andLOC_Os07g23430) appeared to be expressed at low levels and primarily inthe leaf. The analysis of genes encoding the FatB isoforms showed thatFatB-1 (Genbank identifier AP000399, Tigr LOC_Os06g05130) and FatB-2(Genbank identifier AC108870, TIGR identifier Os11g43820) were morehighly expressed in the grain than the other two genes. The Tos-17transpositional insertion mutant had an insertion in the gene encodingAP000399.

In leaf tissue, FatB-1 (Genbank identifier AP000399, TIGRLOC_Os06g05130) and FatB-4 (AP004236, TIGR LOC_Os06g39520) were morehighly expressed, referring to the relative abundance of mRNAtranscripts from the genes. When compared to the abundance of thetubulin gene transcripts as a standard, which was a moderately abundantmRNA in wheat grain and was therefore expected to similarly abundant inrice grain, all the FatB and Fad2 mRNAs accumulated to low levels asdetermined by RT-PCR. However, this conclusion was based on theassumption that the primers, for each of the genes tested, hybridized tothe target transcripts with similar efficiency to the tubulinprimers/transcript.

This conclusion is corroborated by EST database searching. When theFad2-1 (TIGR Os02g48560) sequence was used to search rice EST sequences,transcript sequences were present in panicle, root and whole plant cDNAlibraries. In contrast, Fad2-2 (TIGR Os07g23410) and Fad2-3 (TIGROs07g23430) sequences were present only in leaf, shoot or whole plantlibraries. The sequence for Os07g23390, the Fad2-like gene considered tobe a pseudogene, was not present in any of the EST libraries. Similarly,FatB-1 (TIGR Os06g05130) sequences were present in both leaf and panicleEST libraries, whereas FatB-2 sequences (TIGR Os11g43820) were presentonly in panicle or whole plant EST libraries and FatB-1 (TIGROs06g39520) only in a leaf library. FatB-3 (AP005291, Os02g430900)sequences although expressed at a relatively low level to the others inboth leaf and grain as judged by RT-PCR, were present in panicle andwhole plant EST libraries. These searches used the BLAST program on theNCBI website (www.ncbi.nlm.nih.gov/BLAST/Blast.cgi) using the ESTdatabase and limiting the search to sequences from rice.

Based on these data, Fad2-1 and FatB-2 were considered to be the genesthat should be down-regulated for alteration of grain lipid. FatB-3 wasalso thought be important in this regard.

Example 5 Construction of Gene Silencing Constructs and Transformationof Rice

Creation of duplexRNA Construct for Inhibition of Fad2 Expression

A construct was designed to express a duplex RNA (hairpin RNA) indeveloping rice grain in order to reduce expression of Fad2-1. Bytargeting common regions of the three Fad2 gene sequences, the constructwas designed so that it would be effective for all three of theidentified Fad2 genes in rice grain in order to potentially

maximize the effect on fatty acid composition. To improve silencingefficiency, the construct contained an intron between the sense andantisense portions of the inverted repeat sequences as described bySmith et al. (2000).

A 505 basepair fragment was amplified by PCR from the 5′ end of theFad2-1 gene using the oligonucleotides pFad2-F5′AAAGGATCCTCTAGAGGGAGGAGCAGCAGAAGC-3′ (SEQ ID NO:52) and pFad2-R5′-AAAACTAGTGAATTCTACACGTACGGGGTGTACCA-3′ (SEQ ID NO:53). The PCRproduct was ligated into pGEM-Teasy, transformed into E. coli andcolonies containing the insert identified. The XbaI/EcoRI fragment fromthe plasmid

designated pGEM-T-Fad2, containing the 505 bp Fad2 fragment, was thenligated in the sense direction into the vector ZLRint9_BC3895 (obtainedfrom Zhongyi Li, CSIRO Plant Industry) containing the intron Rint9. ABamHI/SpeI fragment from pGEM-T-Fad2 was then ligated (in the antisenseorientation) into the resultant plasmid so that an intron containinghairpin construct was formed. The BamHI/KpnI fragment containing theFad2-1 intron containing hairpin was then inserted into the samerestriction sites of the pBx17casNOT vector (Zhongyi Li, personalcommunication), containing the Bx17 seed specific promoter containing aNos terminator/polyadenylation sequence so that the silencing gene wouldbe expressed in developing seed in the order (promoter)sense-intron-antisense (terminator). The HindIII/NotI fragmentcontaining the Bx17 promoter and Fad2-1 inverted repeat region was theninserted into the same restriction sites of the binary vector pWBvec8(Wang et al., 1998) that combined a selectable maker gene conferringhygromycin resistance. This vector was then introduced intoAgrobacterium and used for rice transformation as described inExample 1. The duplex RNA construct was designated dsRNA-Fad2-1.Creation of duplexRNA Construct for Inhibition of FatB Expression

A 665-basepair fragment of the rice palmitoyl-ACP thioesterase FatB-1gene (Tigr LOC_Os06g05130) was amplified by PCR using primers with thefollowing sequences: Rte-s1, 5′-AGTCATGGCTGGTTCTCTTGCGGC-3′ (SEQ IDNO:54) and Rte-a1, 5′-ACCATCACCTAAGAGACCAGCAGT-3′ (SEQ ID NO:55). ThisPCR fragment was used to make an inverted repeat construct with one copyof the fragment in the sense orientation and a second copy in theantisense orientation, separated by the intron sequence from the 5′ UTRof the cotton microsomsal ω6-desaturase GhFad2-1 gene (Liu et al.,1999). The inverted repeat construct was subsequently inserted into theSacI site between the Ubil promoter and Nos terminator of pUbilcasNOT(from Zhongyi Li based on sequence described in Li et al., 1997). Theinverted repeat portion of rice FatB with the pUbil promoter was theninserted in the NotI site of the binary vector pWBVec8 and introducedinto Agrobacterium as for the Fad2 construct described above. The duplexRNA construct was designated dsRNA-FatB-1.

Analysis of Fatty Acid Composition in Transformed Rice

Ten independent fertile transgenic plants obtained with dsRNA-Fad2-1were tested for the presence of the dRNA gene by PCR using one primercorresponding to a site within the promoter region and a second primerfor the end of the Fad2 sequence. Nine of the ten plants were found tobe positive in the PCR reaction and therefore contained the Fad2 RNAiconstruct. Similarly, 23 fertile transgenic lines were tested for theFatB RNAi construct and 20 lines were found to be PCR positive.

To analyse the effect of the transgenes on fatty acid composition, totallipid was isolated from grain and leaf samples of the transformed riceplants. This was also done for a rice mutant line containing a Tos17insertional sequence in the gene for FatB-1 (TIGR locus Os06g5130corresponding to the protein identified by the Accession No. AP000399)to compare the effect of specifically inactivating this gene. Fatty acidcomposition was determined for each lipid extract by GC-FAME asdescribed in Example 1. The data are presented in Tables 5 to 7 and someof that data is presented graphically in FIGS. 11 to 13. The proportionof each fatty acid was expressed as a percentage of the total fatty acidin the seed oil of the grain as determined by HPLC as described inExample 1.

The most striking and surprising aspect of the results was the extent ofthe change in grain oleic acid and linoleic acid composition in the Fad2dsRNA plants. The proportion of oleic acid in some lines increased from36% to at least 65% (w/w) and that of linoleic acid decreased from 37%to about 13% in the rice line most affected (Line 22A). Surprisingly,the proportion of palmitic acid in the Fad2 lines was also decreased,for example in Line 22A to less than 14% as compared to the control(18%).

In the FatB dsRNA lines, the reduction in the proportion of palmiticacid in the grain was correlated with an increase in the linoleic acidcontent while the proportions of oleic acid and linolenic acids wereessentially unchanged. It was noted that the extent, of the decrease inthe proportion of palmitic acid in both Fad2 and FatB transgenic lineswas similar, but the extent of increase in the linoleic acid level inthe FatB lines was much less than the extent of the decrease observed inthe Fad2 lines. That is, the extent of the change in levels of linoleicwas greater with the Fad2 construct.

An interesting insight into the FatB catalysed step of the pathway isprovided by analysis of the Tos-17 insertional knockout of one isoformof FatB, FatB-1. In the Tos-17 mutant, the extent of the change in theproportions of palmitic acid and oleic acid was reduced compared to theFatB dsRNA lines. These results indicated that genes encoding the FatBisoforms differed in their function.

When the results of the proportions of linoleic acid versus oleic acidwere plotted as a scatter plot (FIG. 14), it was clear that therelationship between these two fatty acids in the Fad2 knockouts was thesame as in wildtype rice, although the proportion of linoleic acid wasvastly reduced. In contrast, with the FatB knockout and

TABLE 5 GC analysis of fatty acid composition in rice grain of FatB andFad2 mutants Lino- Lino- Myristic Palmitic Stearic Oleic leic lenicGrain- Mutant (C14:0) (C16:0) (C18:0) (C18:1) (C18:2) (C18:3) Wild type0.70 18.83 1.64 38.41 36.42 1.29 (WH12) FatB Tos17 0.72 15.13 2.33 37.1538.61 1.87 insertional mutant FatB dsRNA 0.58 11.43 1.81 34.92 46.601.91 transformed line Fad2 dsRNA 0.58 14.26 2.11 64.94 12.62 1.23transformed line Control for 0.85 17.91 2.60 33.23 39.84 1.76 Tos17Control for 1.03 18.86 1.84 34.08 39.63 1.75 FatB dsRNA Control for 1.0217.47 2.38 36.03 37.42 1.50 Fad2 dsRNA

TABLE 6 GC analysis of fatty acid composition in rice leaves of FatB andFad2 mutants Lauric Myristic Palmitic Stearic Oleic Linoleic LinolenicLeaf- mutant (C12:0) (C14:0) (C16:0) (C18:0) (C18:1) (C18:2) (C18:3)Wild-type (WF2) 0.80 14.43 1.90 2.32 8.99 68.96 FatB Tos17 0.34 11.551.84 2.24 15.04 67.70 insertional mutant FatB dsRNA 0.56 1.01 14.39 2.092.07 13.21 64.56 transformed line Fad2 dsRNA transformed line Controlfor Tos17 0.39 10.52 1.80 2.30 15.61 67.65 Control for FatB 0.72 1.1513.30 2.03 4.04 24.62 52.28 dsRNA

TABLE 7 Relative amounts of grain fatty acids in mutant lines comparedto corresponding control lines (% in mutant/% in corresponding control)Myristic Palmitic Stearic Oleic Linoleic Linolenic (C14:0) (C16:0)(C18:0) (C18:1) (C18:2) (C18:3) Grain fatty acids FatB Tos17 0.85 0.8451 * 0.899 1.118 * 0.9691  1.06 insertional mutant FatB dsRNA 0.57 *0.6060 * 0.980 1.025  1.176 * 1.09 transformed line Fad2 dsRNA  0.570 *0.8167 *  0.887 * 1.802 *  0.3373 *   0.822 * transformed line Leaffatty acids FatB Tos17 nd 0.911 *  0.977 1.03   1.037   0.9992insertional mutant FatB dsRNA nd 0.9247  0.969 1.95 *  1.864 *   0.8098 * transformed line * Statistically significant changeto a lesser extent in the FatB Tos-17 line, although the relationshipbetween linoleic acid and oleic was similar, it is shifted by aconstant. This meant that there was more linoleic acid in the FatBknockout plants for a given amount of oleic acid than in wild-type orFad2 knockout plants. This was consistent with the idea that knockout ofFatB influenced the pathways controlling the amounts of oleic andlinoleic acid but not the step directly linking oleic acid and linoleicacid which was controlled by Fad2.

The results of the proportion of linoleic acid versus palmitic were alsoplotted for all of the rice lines analysed. A positive linearrelationship was observed for the Fad2 knockout lines, inverse of whatwas observed for linoleic versus oleic acid. For the FatB lines,however, a different relationship is observed (FIG. 15). A difference inthe relationship between oleic and palmitin acid was also observed whenthe Fad2 dsRNA plants and FatB dsRNA plants were plotted as a scatterplot (FIG. 16). These results confirmed that the relationship betweenpalmitic and linoleic acid (and oleic acid) was different for the twoperturbations of the pathway.

Principal component analysis of the proportions of the various fattyacids under different perturbations of the pathway confirm thatprincipal component 1 (which indicates the axes that contribute thegreatest variation) was composed of the proportions of linoleic versusoleic whereas the principal component 2 (the second most important setof axes) was composed of proportions of linoleic and oleic acid versuspalmitin acid). This is illustrated in FIG. 17.

We also concluded from the results presented in this Example thatcrossing the dsRNA Fad2 plants with the dsRNA FatB plants or Tos-17 FatBplants to combine the mutations would further increase the relativeproportion of oleic acid and further decrease palmitin and linoleic acidlevels.

Example 6 Production and Use of Antibodies

Antibodies were raised by synthesizing 15 or 16-mer peptides that werepresent in the deduced sequence of FatB. The peptides used to raiseantibodies against FatB were:

FatB-U1 Ac-CGMNKNTRRLSKMPDE (SEQ ID NO:56). This corresponds to thetranslated sequence of FatB (Accession No. AP000399) from amino acidposition number 259 and was also found in sequences translated fromAP005291, ACI08870 and AC004236. However, the sequence was onlyidentical between the four isoforms in the sequence TRRLSKMPDE (SEQ IDNO:57).FatB-U2. Ac-CGEKQWTLLDWKPKKPD (SEQ ID NO:58). This sequence was found inthe translated sequence of all four FatB isoforms.FatB-99. Ac-CGAQGEGNMGFFPAES (SEQ ID NO:59). This sequence was foundonly in AP00399 and was at the very C terminus of the deducedpolypeptide.

After synthesis the peptides were coupled to either Ovalbumin or KeyholeLimpet Haemocyanin protein (KLH) using the cross-linker MBS(maleimidobenzoic acid N-hydroxyl succinimide ester) using standardtechniques. The cross-linked peptide was dialysed and lyophilized andinjected into rabbits at two weekly intervals for two months withFriends incomplete adjuvant at a concentration of approx Antisera raisedagainst FatB-99 detected a clear difference between FatB isoforms inthat a polypeptide of 20 kDa present in wild-type rice was missing inthe Tos-17 mutant line having an insertion in the gene corresponding toTIGR identifier Os06g5130, the product corresponds to FatB-1, thesequence is represented by accession No. AP000399 (FIG. 18). Althoughthis was different to the expected size of approx 40 kDa, such adiscrepancy had been noticed before with FatB isoforms Different size ofFatB products have been observed in developing and mature Cupheawrightii seeds showing five different FatB isoforms, longer size inmature seeds and shorter product in mature seeds (Leonard et al., 1997).

The antisera can be used to detect FatB protein in the transgenic ormutant plant samples and confirm the extent of gene inactivation.

Example 7 Identification of Mutants in Rice Fad2

PCR products spanning the active sites (essentially positions 330 to1020 if the initiating ATG is taken as the start of the numbering inTIGR Loc_Os2g48560) of Fad2-1 (coding sequence corresponding to AP004047 in NCBI database, TIGR locus identifier LOC_Os02g48560) will beproduced from rice DNA extracted from a large number of different riceaccessions. The size of the products will be up to 800 bp depending onthe primers used. Two sets of overlapping primer pairs may need to beused. One possible set of primers is:

Set A (SEQ ID NO: 60) GTGCCGGCGGCAGGATGACGG (positions 5-20 in thealignment shown in FIG. 10) (SEQ ID NO: 61)GCCGACGATGTCGTCGAGCAC (reverse complement ofpositions 379-398 in the alignment shown in FIG. 10) Set 8(SEQ ID NO: 62) TGCCTTCTCCGACTACTCGG (positions 360-379 in FIG. 10)(SEQ ID NO: 63) CCTCGCGCCATGTCGCCTTGG (reverse complement ofpositions 1099-1118 in FIG. 10).

The annealed products will then be subjected to melting in a Rotorgene6000 instrument (Corbett Life Science) or a comparable instrument wheredifferences in melting of heteroduplexes can be sensitively detected bymeans of alteration of fluorescence of a dye LC Green. Hundreds andpossibly thousands of rice lines can be screened daily by thistechnique.

The PCR products from samples showing an altered thermal profile will besequenced and the mutations in Fad2 identified. Selected mutations whichinactivate Fad2 can then be crossed into elite rice lines to producerice lines with reduced Fad2 activity and therefore high oleic acid. Iftwo or all of the Fad2 isoforms need to be eliminated then this can beachieved by identifying lines with the required mutations in differentisoforms and combining the mutations in an elite rice line throughmarker assisted breeding. At least the Fad2-1 gene needs to be inactivefor substantial increases in oleic acid content or decreased linoleicacid content. Inactivation of one or more of the additional Fad2 geneswill further increase oleic acid content and decrease linoleic acidcontent Mutants having mutations in additional Fad2 genes may beidentified in the same way as for Fad2-1, using specific primers for theadditional Fad genes.

Example 8 Stability Analysis—Detection of Hexanal Production in Storage

Experiments are underway with FSA, Werribee to detect the production ofhexanal on storage in wildtype rice. This involves GC using a sampler todetect the volatiles in the headspace of grain stored at hightemperature (40° C.). Once the system is optimized (and also we havesufficient quantity of grain) we will undertake a comparison of theproduction of volatiles, particularly hexanal, upon storage of wildtypeand Fad2 RNAi and FatB RNAi rice lines and suitable combinations ofgenotypes. This is an important quality issue in the rice industry forstorage of grain and also of storage of bran. The production anddetection of other volatiles, which could have a role in affecting grainquality, is also being investigated, both with FSA, Werribee and CSIROEntomology.

Around 10 g of raw brown rice is required for headspace analysis. Thebrown rice is stored at 4° C. (control) and 35° C. for 8 weeks. The gassample released from brown rice can be obtained in the headspace of avial by either heating at 80° C. or by natural diffusion. The volatilecomponents in the headspace can then be analysed by direct injectioninto a GC or GC-MS machine and analysis of the gas chromatographicprofiles (Suzuki et al, 1999). Another method for analysis of volatilesin the headspace is through the trapping of volatiles onto a suitablematrix (eg 250 mg Tenax GR) as described by Nielsen et al. (2004) usingnitrogen as a purge gas. The desorption of the aroma compounds is thendone thermally and the trapped molecules are analysed by GC andidentified using standards.

The expectation that storage of rice would be improved in rice lineswith low linoleic acid is based on a number of observations. Suzuki etal. (1999). have presented data that the amount of free linoleic acidincreases during storage and that the amount of volatile aldehydes suchas pentanal, hexanal and pentanol increase three fold at 35 C. Thecorrelation of hexanal and volatile aldehydes with odour has been notedby other authors as well. On the other hand, Zhou et al. (2002) found areduction of total linoleic acid upon storage and related this to thedecomposition of linoleic acid to other products, including volatilesresponsible for off-odours. The differences in the results may be due todifferences in the extraction and analytical methods.

The production of hexanal for linoleic acid in vitro has beendemonstrated by Nielsen et al. (2004).

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

This application claims priority from AU 2006903810, the entire contentsof which are incorporated herein by reference.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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1-81. (canceled)
 82. A method of preparing food, the method comprisingcooking an edible substance in rice oil from rice grain having a fattyacid composition comprising greater than 60% w/w oleic acid, less than15% w/w palmitic acid and less than 25% w/w linoleic acid, wherein therice oil has not been exposed to a procedure which alters the ratio ofoleic acid to linoleic acid, palmitic acid to oleic acid, and/orpalmitic acid to linoleic acid when compared to the ratio in the ricegrain.
 83. The method of claim 82, wherein in the rice oil the weightratio of oleic acid to linoleic acid is greater than 3.0:1.
 84. Themethod of claim 82, wherein the rice oil has a fatty acid compositioncomprising greater than 60% oleic acid, less than 12% palmitic acid,and/or less than 15% linoleic acid.
 85. The method of claim 82, whereinthe rice oil further comprises γ-oryzanol.
 86. The method of claim 82,wherein the rice oil is substantially purified.
 87. The method of claim86, wherein the substantially purified rice oil is at least 75% freefrom other components with which it is naturally associated.
 88. Themethod of claim 87, wherein the substantially purified rice oil is atleast 90% free from other components with which it is naturallyassociated.